Induce and enhance immune responses using recombinant replicon systems

ABSTRACT

The present disclosure generally relates to the use of different self-amplifying RNA molecules to enhance immune responses, for example immune responses following prophylactic vaccination or therapeutic administration. Some embodiments relate to compositions and methods for inducing an immune response in a subject using prime-boost immunization regimens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. § 119(e) ofU.S. Ser. No. 62/619,540, filed Jan. 19, 2018, the entire contents ofwhich is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The material in the accompanying sequence listing is hereby incorporatedby reference into this application. The accompanying sequence listingtext file, name SGI2230_1WO_Sequence_Listing.txt, was created on Jan.16, 2019, and is 3 kb. The file can be accessed using Microsoft Word ona computer that uses Windows OS.

FIELD

Aspects of the present disclosure relate to the field of virology,infectious diseases, and immunology. More particularly, the disclosurerelates to compositions and methods for inducing and/or enhancing animmune response in a subject by the sequential administration of atleast two immunogenic compositions comprising different RNA replicons.

BACKGROUND

Generating a large population of antigen-specific memory CD8 T cells isa desirable goal for vaccine design against a variety of animal andhuman diseases. Numerous studies performed on experimental models havedemonstrated that the overall number of antigen-specific memory CD8 Tcells present at the time of infection correlates strongly with theability to confer host protection against a range of differentpathogens. Currently, one the most conceivable approaches to rapidlygenerate a large population of memory CD8 T cells is through the use ofprime-boost vaccination. Indeed, multi-dose immunizations, for therapyor for disease prevention, have been reported to be often more effectivethan single-dose immunizations. This difference has been observed fordifferent types of vaccines, including live attenuated vaccines,inactivated vaccines, recombinant protein subunit vaccines, andpolysaccharide vaccines. There is still a need for more effectiveheterologous prime-boost regimes.

SUMMARY

This section provides a general summary of the present application andis not comprehensive of its full scope or all of its features.

The present disclosure provides compositions and methods for deliveringtwo RNA replicons into a subject for various applications. In someembodiments, the compositions and methods disclosed herein allow forinducing and/or enhancing an immune response in the subject. In someembodiments, the compositions and methods disclosed herein can be usedfor the production of a molecule of interest, such as, for example, atherapeutic polypeptide in the subject.

In one aspect, some embodiments disclosed herein relate to a method forinducing an immune response in a subject, the method includesadministering to the subject at least one dose of a priming compositioncomprising a first RNA replicon which encodes a first antigen; andsubsequently administering to the subject at least one dose of aboosting composition comprising a second RNA replicon which encodes asecond antigen, wherein the first and second RNA replicons are differentfrom each other. The first and second RNA replicons can be any describedherein.

In another aspect, some embodiments disclosed herein relate to a methodfor delivering two RNA replicons into a subject, the method includesadministering to the subject a first nucleic acid sequence encoding afirst RNA replicon which encodes a first antigen; and subsequentlyadministering to the subject a second nucleic acid sequence encoding asecond RNA replicon which encodes a second antigen, wherein the firstand second RNA replicons are different from each other. The first andsecond RNA replicons can be any described herein.

Implementations of embodiments of the methods according to the presentdisclosure can include one or more of the following features. In someembodiments, the first antigen and the second antigen are identical toeach other. In some embodiments, amino acid sequences of the first andthe second antigens are homologous to each other. In some embodiments,the amino acid sequence of the first antigen exhibits at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acidsequence of the second antigen. In some embodiments, the first and thesecond antigens comprise at least one cross-reactive antigenicdeterminant. In some embodiments, the first and the second antigensinduce substantially the same immune response in the subject. In someembodiments, the first RNA replicon is capable of activating an immunesystem of the subject through at least one immunological mechanism thatis different from an immunological mechanism by which the immune systemis capable of being activated by the second RNA replicon. In someembodiments, the at least one immunological mechanism is selected fromthe group consisting of differential activation of protein kinase R(PKR), retinoic acid-inducible gene I (RIG-I), autophagy pathways,Toll-like receptors (TLRs), stress granules, RNase R, and oligoadenylatesynthetases (OAS).

In some embodiments disclosed herein, at least one of the first andsecond RNA replicons is a modified RNA replicon. In some embodiments, atleast one of the first and second RNA replicons is derived from apositive-strand RNA virus. In some embodiments, at least one of thefirst and second RNA replicons is derived from a virus species belongingto a family selected from the group consisting of Togaviridae family,Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family,Arteroviridae family, Picornaviridae family, Astroviridae family,Coronaviridae family, and Paramyxoviridae family. In some embodiments,at least one of the first and second RNA replicons is derived from anAlphavirus or an Arterivirus. In some embodiments, at least one of thefirst and second RNA replicons is derived from an alphavirus speciesselected from the group consisting of Eastern equine encephalitis virus(EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus(EVEV), Mucambo virus (MUCV), Semliki forest virus (SFV), Pixuna virus(PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV),O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus(BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV),Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus(AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus(KYZV), Western equine encephalitis virus (WEEV), Highland J virus(HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), Salmonid alphavirus(SAV), and Buggy Creek virus (BCRV).

In some embodiments disclosed herein, both of the first and second RNAreplicons are derived from alphavirus species. In some embodiments, thefirst and second RNA replicons are derived from the same alphavirusspecies or from two different alphavirus species. In some embodiments,the first RNA replicon is derived from an alphavirus and the second RNAreplicon is derived from a non-alphavirus species. In other embodimentsthe first RNA replicon is derived from a non-alphavirus and the secondRNA replicon is derived from an alphavirus species. In some embodimentsthe first RNA replicon is derived from an Arterivirus (e.g. EAV) and thesecond RNA replicon is derived from an alphavirus (e.g. VEEV). In someembodiments, at least one of the first and second RNA repliconscomprises a modified 5′-UTR with one or more nucleotide substitutions atposition 1, 2, 4, or a combination thereof. In some embodiments, atleast one of the one or more nucleotide substitutions is a nucleotidesubstitution at position 2 of the modified 5′-UTR. In some embodiments,the nucleotide substitution at position 2 of the modified 5′-UTR is aU→G substitution.

In some embodiments, at least one of the first and second RNA repliconsis a modified RNA replicon comprising a modified 5′-UTR and is devoid ofat least a portion of a nucleic acid sequence encoding one or more viralstructural proteins. In some embodiments, the modified RNA replicon isdevoid of a substantial portion of the nucleic acid sequence encodingone or more viral structural proteins. In some embodiments, the modifiedRNA replicon comprises no nucleic acid sequence encoding viralstructural proteins. In some embodiments, at least one of the first andsecond RNA replicons is a modified alphavirus replicon comprising one ormore RNA stem-loops in a structural element of a viral capsid enhancer.In some embodiments, at least one of the first and second RNA repliconsis a modified alphavirus replicon comprising coding sequence for aheterologous non-structural protein nsP3. In some embodiments, theheterologous non-structural protein nsP3 is a Chikungunya virus (CHIKV)nsP3 or a Sindbis virus (SINV) nsP3. In some embodiments, at least oneof the first and second antigens is expressed under control of a 26Ssubgenomic promoter or a variant thereof. In some embodiments, the 26Ssubgenomic promoter is a SINV 26S subgenomic promoter, RRV 26Ssubgenomic promoter, or a variant thereof.

In some embodiments disclosed herein, at least one of the first andsecond RNA replicons is derived from an arterivirus species selectedfrom the group consisting of Equine arteritis virus (EAV), Porcinerespiratory and reproductive syndrome virus (PRRSV), Lactatedehydrogenase elevating virus (LDV), and Simian hemorrhagic fever virus(SHFV). In some embodiments, both of the first and second RNA repliconsare derived from arterivirus species. In some embodiments, the first andsecond RNA replicons are derived from the same arterivirus species orfrom two different arterivirus species. In some embodiments, the firstRNA replicon is derived from an arterivirus, and the second RNA repliconis derived from a non-arterivirus species. In some embodiments, thefirst RNA replicon is derived from an arterivirus and the second RNAreplicon is derived from an alphavirus. In some embodiments, the firstRNA replicon is an unmodified RNA replicon derived from an arterivirusspecies. In some embodiments, the first RNA replicon is a modified RNAreplicon derived from an arterivirus species. In some embodiments, thefirst RNA replicon is an RNA replicon derived from an alphavirus speciesand the second RNA replicon is an RNA replicon derived from anarterivirus species. In some embodiments, the first RNA replicon is anunmodified RNA replicon derived from an alphavirus species. In someembodiments, the first RNA replicon is a modified RNA replicon derivedfrom an alphavirus species.

In some embodiments disclosed herein, the method according to thisaspect and other aspects of the disclosure further includes one or moresubsequent boosting steps, e.g., one or more subsequent administrationsof the boosting composition. In some embodiments, one or more of thepriming composition and the boosting composition further comprises apharmaceutically acceptable carrier. In some embodiments, the subject isan aquatic animal. In some embodiments, the subject is an avian species,a crustacean species, or a fish species. In some embodiments, the avianspecies is an avian species for food consumption. In some embodiments,the crustaceans are shrimp. In some embodiments, the fish species is afish species used in aquaculture. In some embodiments, the subject is amammal. In some embodiments, the mammal is human, horse, pig, primate,mouse, ferret, rat, cotton rat, cattle, swine, sheep, rabbit, cat, dog,goat, donkey, hamster, or buffalo.

In one aspect, some embodiments disclosed herein relate to a compositionwhich comprises: a priming composition comprising a first RNA repliconwhich encodes a first antigen; and a boosting composition comprising asecond RNA replicon which encodes a second antigen, wherein the firstand second RNA replicons are different from each other.

In another aspect, some embodiments disclosed herein relate to acomposition which comprises: a first nucleic acid sequence encoding afirst RNA replicon which encodes a first antigen; and a second nucleicacid sequence encoding a second RNA replicon which encodes a secondantigen, wherein the first and second RNA replicons are different fromeach other, wherein the first replicon and the second replicon comprisesat least one expression cassette comprising a promoter operably linkedto a coding sequence for a molecule of interest. The first RNA repliconand second RNA replicon can be any described herein.

Implementations of embodiments of the compositions according to theabove aspects of the present disclosure can include one or more of thefollowing features. In some embodiments, the first and the secondantigens are identical to each other. In some embodiments, amino acidsequences of the first and the second antigens are homologous to eachother. In some embodiments, the amino acid sequence of the first antigenexhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the amino acid sequence of the second antigen. In someembodiments, the first and the second antigens comprise at least onecross-reactive antigenic determinant. In some embodiments, the first andthe second antigens induce substantially the same immune response in thesubject. In some embodiments, the first RNA replicon can activate animmune system of the subject through at least one immunologicalmechanism that is different from an immunological mechanism by which theimmune system can be activated by the second RNA replicon. In someembodiments, the at least one immunological mechanism is selected fromthe group consisting of differential activation of protein kinase R(PKR), retinoic acid-inducible gene I (RIG-I), autophagy pathways,Toll-like receptors (TLRs), stress granules, RNase R, and oligoadenylatesynthetases (OAS).

In some embodiments disclosed herein, at least one of the first andsecond RNA replicons is a modified replicon. In some embodiments, atleast one of the first and second RNA replicons is derived from apositive-strand RNA virus. In some embodiments, at least one of thefirst and second RNA replicons is derived from a virus species belongingto a family selected from the group consisting of Togaviridae family,Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family,Arteroviridae family, Picornaviridae family, Astroviridae family,Coronaviridae family, and Paramyxoviridae family. In some embodiments,at least one of the first and second RNA replicons is derived from anAlphavirus or an Arterivirus. In some embodiments, at least one of thefirst and second RNA replicons is derived from an alphavirus speciesselected from the group consisting of any one or more of: Eastern equineencephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV),Everglades virus (EVEV), Mucambo virus (MUCV), Semliki forest virus(SFV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus(CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), BarmahForest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaruvirus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus(SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV),Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV),Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV),Salmonid alphavirus (SAV), and Buggy Creek virus (BCRV), or from a groupconsisting of every possible combination or sub-combination of theviruses. For example in some embodiments the alphavirus species can beselected from the group consisting of Eastern equine encephalitis virus(EEEV), Venezuelan equine encephalitis virus (VEEV), and Evergladesvirus (EVEV).

In some embodiments disclosed herein, both of the first and second RNAreplicons are derived from alphavirus species. In some embodiments, thefirst and second RNA replicons are derived from the same alphavirusspecies or from two different alphavirus species. In some embodiments,the first RNA replicon is derived from an alphavirus and the second RNAreplicon is derived from a non-alphavirus species. In some embodiments,at least one of the first and second RNA replicons comprises a modified5′-UTR with one or more nucleotide substitutions at position 1, 2, 4, ora combination thereof. In some embodiments, at least one of the one ormore nucleotide substitutions is a nucleotide substitution at position 2of the modified 5′-UTR. In some embodiments, the nucleotide substitutionat position 2 of the modified 5′-UTR is a U→G substitution.

In some embodiments, at least one of the first and second RNA repliconsis a modified RNA replicon comprising a modified 5′-UTR and is devoid ofat least a portion of a nucleic acid sequence encoding one or more viralstructural proteins. In some embodiments, the modified RNA replicon isdevoid of a substantial portion of the nucleic acid sequence encodingone or more viral structural proteins. In some embodiments, the modifiedRNA replicon comprises no nucleic acid sequence encoding viralstructural proteins. In some embodiments, at least one of the first andsecond RNA replicons is a modified alphavirus replicon comprising one ormore RNA stem-loops in a structural element of a viral capsid enhanceror a variant thereof. In some embodiments, at least one of the first andsecond RNA replicons is a modified alphavirus replicon comprising acoding sequence for a heterologous non-structural protein nsP3. In someembodiments, the heterologous non-structural protein nsP3 is aChikungunya virus (CHIKV) nsP3, a Sindbis virus (SINV) nsP3, or avariant thereof. In some embodiments, at least one of the first andsecond antigens is expressed under control of a 26S subgenomic promoteror a variant thereof. In some embodiments, the 26S subgenomic promoteris a SINV 26S subgenomic promoter, RRV 26S subgenomic promoter, or avariant thereof.

In some embodiments disclosed herein, at least one of the first andsecond RNA replicons is derived from an arterivirus species selectedfrom the group consisting of Equine arteritis virus (EAV), Porcinerespiratory and reproductive syndrome virus (PRRSV), Lactatedehydrogenase elevating virus (LDV), and Simian hemorrhagic fever virus(SHFV). In some embodiments, both of the first and second RNA repliconsare derived from an arterivirus species. In some embodiments, the firstand second RNA replicons are derived from the same arterivirus speciesor from two different arterivirus species. In some embodiments, thefirst RNA replicon is derived from an arterivirus, and the second RNAreplicon is derived from a non-arterivirus species. In some embodiments,the first RNA replicon is derived from an arterivirus and the second RNAreplicon is derived from an alphavirus. In some embodiments, the firstRNA replicon is an unmodified RNA replicon derived from an arterivirusspecies. In some embodiments, the first RNA replicon is a modified RNAreplicon derived from an arterivirus species. In some embodiments, thefirst RNA replicon is an RNA replicon derived from an alphavirus speciesand the second RNA replicon is an RNA replicon derived from anarterivirus species. In some embodiments, the first RNA replicon is anunmodified RNA replicon derived from an alphavirus species. In someembodiments, the first RNA replicon is a modified RNA replicon derivedfrom an alphavirus species.

In some embodiments disclosed herein, the compositions according to thepresent disclosure further comprise compositions for one or moresubsequent boosting steps, e.g., one or more subsequent administrationsof the boosting composition. In some embodiments, one or more of thepriming composition and the boosting composition further comprises apharmaceutically acceptable carrier. In some embodiments, the subject isa mammal. In some embodiments, the mammal is human, horse, pig, primate,mouse, ferret, rat, cotton rat, cattle, swine, sheep, rabbit, cat, dog,goat, donkey, hamster, or buffalo.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative embodiments andfeatures described herein, further aspects, embodiments, objects andfeatures of the application will become fully apparent from thedrawings, the detailed description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a non-limiting example of a methodof inducing an immune response in a subject in accordance with someembodiments of the disclosure. In this example, the magnitude of immuneresponse, as determined by total number of antigen specific CD8 T cells,to a traditional prime-boost regimen (dashed line) or to a heterologousprime-boost regimen (solid line) is plotted over time.

FIGS. 2A-2B schematically summarize the results of experiments performedto analyze immune responses in mice after various prime-boostingschedules in accordance with some embodiments of the disclosure. In FIG.2A, mean frequencies of effector IFN-γ-secreting CD8+ T cell responseswere determined by enzyme-linked immunospot (ELISpot) assays onsplenocytes derived from immunized BALB/c mice 14 days after boost (astands for alphavirus replicon). In FIG. 2B, geometric means of totalIgG titers (inverse of ED20%) at 14 days after boost were determined byenzyme-linked immunosorbent assays (ELISA). All immune responses areshown with 95% confidence intervals and statistics displayed are usingnon-parametric unpaired Mann-Whitney test.

The foregoing and other features of the present disclosure will becomemore fully apparent from the following description and appended claims,taken in conjunction with the accompanying drawings. Understanding thatthese drawings depict only several embodiments in accordance with thedisclosure and are not to be considered limiting of its scope, thedisclosure will be described with additional specificity and detailthrough use of the accompanying drawings.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure generally relates to the use of differentself-amplifying mRNA molecules to enhance immune responses, for exampleimmune responses following prophylactic vaccination and/or therapeuticadministration. Some embodiments of the disclosure relate tocompositions and methods for inducing an immune response in a subjectusing heterologous prime-boost immunization regimens that can be usedprophylactically and/or therapeutically. In some embodiments, thecompositions and methods disclosed herein can be deployed for theproduction of a molecule of interest, e.g., a therapeutic polypeptide,in a subject.

Generating a large population of antigen-specific memory CD8 T cells isa desirable goal for vaccine design against a variety of animal andhuman diseases. One approach to efficaciously generate a largepopulation of memory CD8 T cells is through the use of prime-boostvaccination in a “heterologous” prime-boost format, which involvespriming the generation of memory CD8 T cells with an antigen deliveredin one vector and then administering the same antigen, or essentiallythe same antigen, in the context of a different vector at a later timepoint.

Some embodiments disclosed herein relate to heterologous prime-boostregimens that involve sequential administrations of the same immunogenusing two different modalities as a strategy to elicit superior immuneresponses in subjects. This strategy can be employed for a variety ofchallenging pathogens including, but are not limited to, malaria, HIV,TB, and Ebola. Heterologous prime-boosts can result in superior memoryresponses, a higher magnitude of CD8+ T cell responses, a broadening ofT cell epitopes recognized by the immune system, and an increase inpolyfunctionality of T cells. Alphavirus-derived replicons (for example,Sindbis, VEE, and Semliki-forest virus) have been employed inheterologous prime-boost settings in combination with protein, DNA, andvirosomes. These have proven to be effective in small animal models inmice for human papillomavirus (HPV) and human immunodeficiency virus(HIV), as well as in non-human primates (NHPs) with the replicon beingdelivered in the particle form for Dengue. Furthermore, fully syntheticAlphavirus-derived replicons have been used extensively in homologousprime-boost regimes. The invention provides regimens having twoimmunologically different replicons for use in heterologous prime-boostregimens. The invention also provides regimens having repeatedadministration of Alphavirus-derived replicons expressing therapeuticproteins that can employ either homologous or heterologousadministration regimens.

As disclosed herein, by priming and boosting the generation of memoryCD8 T cells with two immunologically different RNA replicons, immuneresponses can be strategically improved to tackle more complexpathogens. For example, with regards to vaccines, recall responses canbe negatively impacted by pre-existing antibodies or T cell responses tothe platform delivering the antigen (anti-vector immunity). Similarly,multiple administrations of the antigen using the same platformstimulates the immune system in the exact same way, but may be moreinherently self-limiting due to its inability to synergize withalternate mechanisms of immune detection. Without being bound to anyparticular theory, it is believed that heterologous prime-boostimmunization functions to circumvent the first problem since it can bedesigned to bypass pre-existing antibodies or T cells responsesdepending on the causative mechanism of reduced responses. Furthermore,heterologous prime-boosts using two immunologically different repliconscan be engineered so that the follow-on administrations activate theimmune system in different ways that synergize with the initialadministration.

In another example, with regards to therapeutics, recall response canreduce the duration and magnitude of heterologous protein expression asimmune responses directed against the vector can result in the clearanceof cells expressing the therapeutic protein. Without being bound to anyparticular theory, it is believed that heterologous prime-boostimmunization bypasses this issue by reducing the ability for the immunesystem to recognize the replicon that is expressing the protein uponrepeat administrations, thereby delaying clearance.

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative alternatives described in thedetailed description, drawings, and claims are not meant to be limiting.Other alternatives may be used, and other changes may be made, withoutdeparting from the spirit or scope of the subject matter presented here.It will be readily understood that the aspects, as generally describedherein, and illustrated in the Figures, can be arranged, substituted,combined, and designed in a wide variety of different configurations,all of which are explicitly contemplated and made part of thisapplication.

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisapplication pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art.

Some Definitions

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “a cell”includes one or more cells, comprising mixtures thereof “A and/or B” isused herein to include all of the following alternatives: “A”, “B”, “Aor B”, and “A and B”.

The term “about”, as used herein, has its ordinary meaning ofapproximately. If the degree of approximation is not otherwise clearfrom the context, “about” means either within plus or minus 10% of theprovided value, or rounded to the nearest significant figure, in allcases inclusive of the provided value. Where ranges are provided, theyare inclusive of the boundary values.

The term “antigenic determinant” or “epitope”, as used herein, refers toa part of an antigen (e.g., a polypeptide), for example a part of theprimary, secondary, tertiary, or quaternary structure of the antigen,that is recognized by the immune system, for example antibodies, B cells(e.g., B lymphocytes) and/or T cells. In some embodiments, the antigenicdeterminant is a site on the surface of the antigen. In someembodiments, the antigenic determinant is a site that an antibodymolecule binds to the antigen. The term “cross-reactive antigenicdeterminant” refers to the ability of an antigenic determinant presenton two or more different antigen molecules (e.g., polypeptides) to bebound by the same antibody. Furthermore, it is to be understood that thetwo or more antigen molecules comprising the antigenic determinantcapable of being bound by the same antibody can be, for example, thesame molecules or fragments thereof, variants of one another, ordifferent molecules. By way of example with reference to polypeptidescomprising a cross-reactive antigenic determinant capable of being boundby the same antibody, the polypeptides can have the same or a differentprimary amino acid sequence, however, the polypeptides each comprise anantigenic determinant (e.g., “cross-reactive”) that can be bound by thesame antibody.

The term “derived from” used herein refers to an origin or source, andmay include naturally occurring, recombinant, unpurified or purifiedmolecules. The molecules of the present disclosure may be derived fromviral or non-viral molecules. A protein or polypeptide derived from anoriginal protein or polypeptide may comprise the original protein orpolypeptide, in part or in whole, and may be a fragment or variant ofthe original protein or polypeptide. In some embodiments the RNAreplicon is substantially a viral genome, meaning that the sequencecontains sufficient genetic information for the replicon to autonomouslyreplicate within a host cell or treated organism, but is not a completewild-type viral genome.

The term “gene” is used broadly to refer to any segment of nucleic acidmolecule that encodes a protein or that can be transcribed into afunctional RNA. Genes may include sequences that are transcribed but arenot part of a final, mature, and/or functional RNA transcript, and genesthat encode proteins may further comprise sequences that are transcribedbut not translated, for example, 5′ untranslated regions, 3′untranslated regions, introns, etc. Further, genes may optionallyfurther comprise regulatory sequences required for their expression, andsuch sequences may be, for example, sequences that are not transcribedor translated. Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

By “immune response” or “immunity” as the terms are interchangeably usedherein, is meant the induction of a humoral response (e.g., B cell)and/or cellular response (e.g., T cell). Suitably, a humoral immuneresponse may be assessed by measuring the antigen-specific antibodiespresent in serum of immunized animals in response to introduction of oneor more antigens into the host. In some exemplary embodiments below, theimmune response is assessed by the enzyme-linked immunospot (ELISpot)assays on splenocytes derived from immunized animals, or by theenzyme-linked immunosorbant assay (ELISA) of sera of immunized animals,as discussed in Example 1 below. The term “immunogen” or “immunogenic”refers to a molecule that induces a specific immune response.

The terms “modified” and “sequence modification” as used herein inrelation to nucleic acid molecules, polypeptides, and RNA replicons areintended to define nucleic acid molecules, polypeptides, and RNAreplicons which differ in nucleotide sequence or amino acid sequencefrom the native (e.g., wild-type or unmodified). The terms“naturally-occurring” and “wild-type”, as used herein, refer to a formfound in nature. For example, a naturally occurring, unmodified, orwild-type nucleic acid molecule, nucleotide sequence, RNA replicon, orprotein may be present in and isolated from a natural source, and is notintentionally modified by human manipulation. As described in detailbelow, the nucleic acid molecules, polypeptides, and RNA repliconsaccording to some embodiments of the present disclosure are modifiednucleic acid molecules, polypeptides, and RNA replicons, and thereforethey are non-naturally occurring RNA replicons.

The terms “prime” and “boost” are intended to have their ordinarymeanings in the art. “Priming” refers to immunizing a subject with afirst antigenic composition to induce an immunity of the subject to anantigen that can be recalled upon subsequent exposure(s) to the sameantigen or similar antigen. In some embodiments, priming induces ahigher level of immune response to the antigen upon subsequentimmunization (“boosting”) with the same antigenic composition or with arelated antigenic composition (e.g., a composition comprising an antigenhaving at least one cross-reactive antigenic determinant) than theimmune response level obtained by immunization with a single antigeniccomposition, e.g., the priming composition alone or the boostingcomposition alone. “Booster dose” refers to an administration of anantigenic composition (e.g., a vaccine) after an earlier (prime) dose.After initial immunization (e.g., administration of a primingcomposition) to a subject, in some embodiments, a booster dose can beadministered one or more times to the same subject for re-exposure tothe same immunogenic antigen or an antigen having at least onecross-reactive antigenic determinant with the antigen used in thepriming composition.

The terms “RNA replicon” and “replicon RNA” used interchangeably herein,refer to RNA which contains all of the genetic information required fordirecting its own amplification or self-replication within a permissivecell. To direct its own replication, the RNA molecule 1) encodespolymerase, replicase, or other proteins which may interact with viralor host cell-derived proteins, nucleic acids or ribonucleoproteins tocatalyze the RNA amplification process; and 2) contain cis-acting RNAsequences required for replication and transcription of the subgenomicreplicon-encoded RNA. These sequences may be bound during the process ofreplication to its self-encoded proteins, or non-self-encodedcell-derived proteins, nucleic acids or ribonucleoproteins, or complexesbetween any of these components. In some embodiments, a modified RNAreplicon molecule typically contains the following ordered elements: 5′viral RNA sequence(s) required in cis for replication, sequences codingfor biologically active nonstructural proteins, promoter for thesubgenomic RNA, 3′ viral sequences required in cis for replication, anda polyadenylate tract. Further, the RNA replicon can be a molecule ofpositive polarity, or “message” sense, and the RNA replicon may be oflength different from that of any known, naturally-occurring RNAviruses. In some embodiments of the present disclosure, the RNA repliconcan lack or functionally lack at least one of the sequences of thestructural viral proteins present in wild-type virus genomes. Byfunctionally lack is meant that the structural viral proteins are notpresent in an amount or in a form that permits them to perform theirusual and natural function. In many embodiments the sequences encodingstructural genes can be substituted with one or more heterologoussequences such as, for example, a sequence encoding a gene of interest(GOI). In some embodiments the GOI can be, for example, a sequenceencoding a polypeptide that is an antigen or antigenic determinant tothe subject patient (such as an antigen described herein), an antibody,or a fragment of an antibody. In those instances where the RNA repliconis to be packaged into a recombinant alphavirus particle, it mustcontain one or more sequences, so-called packaging signals, which serveto initiate interactions with alphavirus structural proteins that leadto particle formation. The RNA replicons of the invention can have theability to self-amplify and can self-amplify within a host cell oranimal cell. In various embodiments the RNA replicons can be at least 1kb or at least 2 kb or at least 3 kb or at least 4 kb or at least 5 kbor at least 6 kb or at least 7 kb or at least 8 kb or at least 10 kb orat least 12 kb or at least 15 kb or at least 17 kb or at least 19 kb orat least 20 kb in size, or can be 100 bp-8 kb or 500 bp-8 kb or 500 bp-7kb or 1-7 kb or 1-8 kb or 2-15 kb or 2-20 kb or 5-15 kb or 5-20 kb or7-15 kb or 7-18 kb or 7-20 kb in size. “Fragments” of a molecule (e.g. anucleic acid, polypeptide, or antibody molecule) can contain at least atleast 10 or at least 20 or at least 30 or at least 50 or at least 75 orat least 100 or at least 200 or at least 300 or at least 500 or at least1 kb or at least 2 kb or at least 3 kb or at least 5 kb nucleotides fora nucleic acid, or amino acids for a polypeptide molecule. A fragmentcan also be a binding domain of a specific binding molecule. In someembodiments the RNA replicons are not viral vectors, which utilize viralproteins (e.g. a capsid protein encoded on the viral vector) to deliverits nucleic acid into a host cell. The RNA replicons of the inventioncan lack, functionally lack, or not have a capsid or viral particle, orcan not be encapsidated in a capsid or comprised in a viral particle.

The RNA replicons of the invention can be derived from a naturallyoccurring or wild-type virus (e.g. an RNA virus or retrovirus describedherein), meaning that the replicon has been modified from a wild-typeviral genome. The RNA replicons of the invention can include sequencesnot present in a wild-type viral genome, for example one or moreheterologous sequence(s) (e.g. one or more gene(s) of interest) and/orother sequences or modifications as described herein. The RNA repliconscan also have one or more sequences deleted or functionally deleted froma wild-type genome (e.g. viral structural proteins). A sequence isfunctionally deleted when it is not present in an amount or in a formthat permits it to perform its usual and natural function. For example asequence can be deleted completely or substantially, or otherwiseshortened so that it does not perform its usual and natural function. Indifferent embodiments the RNA replicons of the invention can have atleast at least 50% or at least 60% or at least 70% or at least 80% or atleast 90% or at least 95% or 80-99% or 90-95% or 90-99% or 95-99% or97-99% or 98-99% sequence identity with a sequence of a wild typegenome. In some embodiments the percent of sequence identity can becalculated while not counting one or more heterologous sequence(s) thatmay be present on the replicon (e.g. a gene of interest), and/or notcounting the deletion of one or more sequences that would be naturallypresent in the wild-type genome (e.g. one or more structural genes).

In some embodiments, the RNA replicons disclosed herein are engineered,synthetic, or recombinant RNA replicons. As used herein, the termrecombinant means any molecule (e.g. DNA, RNA, etc.), that is, orresults, however indirect, from human manipulation of a polynucleotide.As non-limiting examples, a cDNA is a recombinant DNA molecule, as isany nucleic acid molecule that has been generated by in vitro polymerasereaction(s), or to which linkers have been attached, or that has beenintegrated into a vector, such as a cloning vector or expression vector.As non-limiting examples, a recombinant RNA replicon can be one or moreof the followings: 1) synthesized or modified in vitro, for example,using chemical or enzymatic techniques (for example, by use of chemicalnucleic acid synthesis, or by use of enzymes for the replication,polymerization, exonucleolytic digestion, endonucleolytic digestion,ligation, reverse transcription, transcription, base modification(including, e.g., methylation), or recombination (including homologousand site-specific recombination) of nucleic acid molecules; 2) conjoinednucleotide sequences that are not conjoined in nature; 3) engineeredusing molecular cloning techniques such that it lacks one or morenucleotides with respect to the naturally occurring nucleotide sequence;and 4) manipulated using molecular cloning techniques such that it hasone or more sequence changes or rearrangements with respect to thenaturally occurring nucleotide sequence.

The term “variant” of a protein used herein refers to a polypeptidehaving an amino acid sequence that is the same or essentially the sameas that of the reference protein except having at least one amino acidmodified, for example, deleted, inserted, or replaced, respectively. Theamino acid replacement may be a conservative amino acid substitution,preferably at a non-essential amino acid residue in the protein. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains areknown in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, and histidine), acidic side chains(e.g., aspartic acid, and glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, andcysteine), non-polar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, and tryptophan),beta-branched side chains (e.g., threonine, valine, and isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, andhistidine). A variant of a protein may have an amino acid sequence atleast about 80%, 90%, 95%, 98%, or 99%, preferably at least about 90%,more preferably at least about 95%, identical to the amino acid sequenceof the protein. Preferably, a variant is a functional variant of aprotein that retains the same function as the protein.

Also of interest of the present disclosure are variants of thepolynucleotides described herein. Such variants may benaturally-occurring, including homologous polynucleotides from the sameor a different species, or may be non-natural variants, for examplepolynucleotides synthesized using chemical synthesis methods, orgenerated using recombinant DNA techniques. With respect to nucleotidesequences, degeneracy of the genetic code provides the possibility tosubstitute at least one base of the protein encoding sequence of a genewith a different base without causing the amino acid sequence of thepolypeptide produced from the gene to be changed. Hence, thepolynucleotides of the present disclosure may also have any basesequence that has been changed from any polynucleotide sequencedisclosed herein by substitution in accordance with degeneracy of thegenetic code. References describing codon usage are readily publiclyavailable. In further embodiments, polynucleotide sequence variants canbe produced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (e.g., changing codons in the viral mRNA to thosepreferred by other organisms such as mammals or fish species).

As will be understood by one of skill in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible sub-rangesand combinations of sub-ranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 articles refers to groupshaving 1, 2, or 3 articles. Similarly, a group having 1-5 articlesrefers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

In some embodiments of the methods or processes described herein, thesteps can be carried out in any order, except when a temporal oroperational sequence is explicitly recited. Furthermore, in someembodiments, the specified steps can be carried out concurrently unlessexplicit claim language recites that they be carried out separately. Forexample, in some embodiments a claimed step of doing X and a claimedstep of doing Y can be conducted simultaneously within a singleoperation, and the resulting process will fall within the literal scopeof the claimed process.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any elements, steps, or ingredients notspecified in the claimed composition or method. As used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claimedcomposition or method. Any recitation herein of the term “comprising”,for example in a description of components of a composition or in adescription of steps of a method, is understood to encompass thosecompositions and methods consisting essentially of and consisting of therecited components or steps.

Headings, e.g., (a), (b), (c), etc., are presented merely for ease ofreading the specification and claims, and do not limit in any way thescope of the disclosure or its alternatives. The use of headings in thespecification or claims does not require the steps or elements beperformed in alphabetical or numerical order or the order in which theyare presented.

Methods for Heterologous Prime-Boost Immunization

Multi-dose immunization, for therapy or for disease prevention, has beenreported to be often more effective than single-dose immunization. It isgenerally believed that generating a high number of antigen-specificmemory CD8 T cells following vaccination is a desirable goal for vaccinedesign against a variety of animal and human diseases, because thisnumber strongly correlates with host immunization and protection. Oneapproach to generate these high numbers of cells is to use a process ofprime-boost immunization, which relies on the re-stimulation ofantigen-specific immune cells following primary memory formation. Insuch a process, there is a “priming” composition which is administeredto the subject first and a “boosting” composition which is subsequentlyadministered one or more times. Without being bound by any particulartheory, it is widely believed that boosting of immune responses byvaccines results in generation of larger numbers of effector cellsrequired for mediating protection against pathogens at the time ofinfection.

Homologous prime-boost immunizations that utilize re-administration ofthe same immunization agent have been used since the initial developmentof vaccines. Classic vaccination approaches relied on a homologousprime-boost regime and have traditionally been unable to elicit immuneresponses strong enough to tackle more challenging diseases. Forexample, although this method is usually effective in boosting thehumoral response to antigen, it has been generally considered to be farless effective at generating increased numbers of CD8 T cells due torapid clearance of the homologous boosting agent by the primed immunesystem, and further fail to boost cellular immunity (CMI).

On the other hand, heterologous prime-boost immunization, or theadministration of the same immunogen using two different modalities, wasrecently developed as a strategy to elicit superior immune responses insubjects. In particular, new vaccine modalities, such as heterologousprime-boosts, have been successfully employed against complex pathogenssuch as malaria, Tuberculosis (TB), human immunodeficiency virus (HIV),and Ebola. Superior memory responses resulting from heterologousprime-boost immunization include, but are not limited to, a highermagnitude of CD8+ T cell responses, a broadening of T cell epitopesrecognized by the immune system, and an increase in polyfunctionality ofT cells. In some embodiments the prime-boost methods of the inventionresult in a significant increase in IFN-γ-secreting CD8+ T cells in thetreated subject. In various embodiments the significant increase can bean increase of at least 25% or at least 50% or at least 100% or at least150% or at least 200% or at least 250% or at least 300% versus singledose administration or versus a homologous prime-boost regimen. Inaddition, a heterologous prime-boost approach is reported to effectivelyboost CMI, especially when vector-based vaccine candidates are used, asit minimizes the interference by anti-vector immunity generated afterpriming. Apart from enhancing the effector cells quantitatively,qualitative differences in secondary memory cells are also seen afterthe boosting. Secondary memory CD8 T cells, in contrast to primarymemory cells, traffic much more efficiently to peripheral tissues andexhibit enhanced cytolysis facilitating effective countering ofpathogens at the site of entry. Additionally, a heterologous prime-booststrategy can result in synergistic enhancement of immune responseresulting in an increased number of antigen-specific T cells, selectiveenrichment of high avidity T cells and increased breadth as well asdepth of the immune response. By way of example, FIG. 1 schematicallydepicts benefits of heterologous prime-boost regimens in that they canresult in improving both the magnitude, length, and the quality of theimmune memory response (figure adapted from Nolz J C and Harty J T, Adv.Exp. Med. Biol., 2011). In this example, booster vaccinations are usedto generate increased numbers of memory CD8 T cells. The magnitude ofimmune response to a traditional prime-boost regimen or to aheterologous prime-boost regimen, as determined by total number ofantigen specific CD8 T cells, is plotted over time. Following primaryvaccine challenge, CD8 T cells undergo expansion, contraction, and forma primary memory population. When this primary memory population of CD8T cells is exposed to a secondary challenge of the same vaccination(homologous boost, dashed line), another round of expansion, contractionand formation of a larger, secondary memory population occurs. Incontrast to a homologous booster vaccination, administration of a CD8 Tcell antigen delivered in the context of a different vector(heterologous boost, solid line) drives greater expansion of the primarymemory CD8 T cells, resulting in a larger secondary memory populationthan what is seen with homologous booster vaccinations.

While heterologous prime-boosts have been reported to increase responsesin certain settings, not all combinations demonstrate improved immunityshowing the importance of determining which combinations are effective.Finding vaccine combinations that elicit broad, durable, andlong-lasting immunity are important for conferring robust protection.More specifically, Alphavirus-derived replicons such as, for example,Sindbis virus, VEE virus, and Semliki-forest virus, have all beenemployed in heterologous prime-boost settings in combination withprotein, DNA, and virosomes. These have proven to be effective inimmunizing small animal models, such as mice, for human papillomavirus(HPV) and human immunodeficiency virus (HIV), as well as in NHPs withthe replicon being delivered in the particle form for Dengue.Furthermore, fully synthetic alphavirus-derived replicons have been usedextensively in homologous prime-boost regimes. Previously, the onlyfully synthetic replicon system that has been widely employed has beenderived from the Alphavirus family of viruses, wherein thenon-structural proteins have been retained and the structural proteinshave been replaced with a gene of interest. However, recent advances inengineering of new replicons have resulted in the production of noveltypes of replicon and have permitted discovering novel vaccinemodalities using only synthetic replicons.

Similarly, classic approaches to therapeutic administration of proteinshave traditionally relied on exogenous injection of proteins in highenough doses to have the desired clinical effect. More recently, nucleicacid or viral-based vectors have been used to deliver a sequence to ahost cell resulting in the expression of a desired protein of interest.However, strictly nucleic acid-based delivery methods such as mRNA,while relatively non-immunogenic, do not have durable and persistentexpression of protein. In contrast, viral-derived methods are capable ofmore durable and persistent protein expression, but are also inherentlyimmunogenic. This can result in immune responses against the cellsproducing the protein, and sometimes the protein itself in the form ofanti-drug antibodies. Viral-based methods of protein delivery also tendto have higher costs and more complex manufacturing, limiting howbroadly this technique can be employed. For this reason, using repliconswith immunologically different mechanisms of activating the immunesystem may allow an increased or more varied number of repeatedinjections allowing for the more persistent expression of therapeuticproteins. This approach would also be expected to help limit theformation of anti-drug antibodies that reduce the level of therapeuticprotein being produced.

In one aspect, various embodiments of the disclosure generally relate tomethods for delivering two RNA replicons into a subject for therapeuticand/or prophylactic applications such as, for example, vaccinationand/or immunization applications. In one aspect, some embodimentsdisclosed herein relate to a method for inducing an immune response in asubject, the method includes administering to the subject at least onedose of a priming composition comprising a first RNA replicon whichencodes a first antigen; and subsequently administering to the subjectat least one dose of a boosting composition comprising a second RNAreplicon which encodes a second antigen, wherein the first and secondRNA replicons are different from each other. In some embodiments, thefirst antigen and the second antigen are identical to each other. Insome embodiments, amino acid sequences of the first and the secondantigens are homologous to each other. In some embodiments the first RNAreplicon and second RNA replicon are derived from genomes of RNA virusesof different genera. In some embodiments, the amino acid sequence of thefirst antigen exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% sequence identity to the amino acid sequence of the second antigen.In some embodiments, the first and the second antigens comprise at leastone cross-reactive antigenic determinant. In some embodiments, the firstand the second antigens induce substantially the same immune response inthe subject.

In some embodiments, the priming composition is administered into thesubject in a single dose. In some embodiments, the priming compositionis administered into the subject in multiple doses. In some embodiments,the boosting composition is administered into the subject in a singledose. In some embodiments, the boosting composition is administered intothe subject in multiple doses. In some embodiments, the primingcomposition and/or the boosting composition is administered to thesubject for at least 2, at least 3, at least 4, at least 5, or at least10 consecutive dosages or any number dosage therebetween. In someembodiments, the priming composition and/or the boosting composition isadministered to the subject for at least 10, at least 12, at least 14,at least 16, or at least 20 consecutive dosages or any number dosagetherebetween. Without being bound to any particular theory, it isgenerally believed that higher antigen doses at priming generally favorthe induction of effector cells, whereas lower doses may preferentiallydrive the induction of immune memory. Hence, higher dose of a primingcomposition, although desirable for immediate responses, may affectdevelopment of memory cells and adversely hamper the effect of highdose. Contrary to the prime dose, higher dose of the boost compositionhas been shown to induce higher magnitude of immune response as thegreater availability of antigen might be driving higher number of memoryB cells into differentiation, thereby amplifying the response. In someembodiments, the priming composition and/or the boosting composition canbe administered into the subject in multiple dosages ranging from about0.001 mg/kg body weight to about 50 mg/kg body weight. This dose rangeis equivalent to about 0.025 μg to 50 μg of RNA replicon in formulatedstate for a 25 g mouse. In some embodiments, preferred doses of thepriming composition and/or the boosting composition comprise less than 1μg of RNA replicon in formulated state. In some embodiments, preferreddoses of the priming composition and/or the boosting compositioncomprise about 100 μg, about 200 μg, about 300 μg, about 400 μg, about500 μg, about 600 μg, about 700 μg of RNA replicon in non-formulatedstate (e.g., naked RNA in saline solution). In various embodimentseither or both of the first and second RNA replicons can be administeredas naked RNA (e.g. in saline) or either or both can be administeredcomprised in a nano-particle; or either one can be administered as nakedRNA and the other administered in a nano-particle. In some embodimentswherein small animal models are involved, the priming composition and/orthe boosting composition can be administered into the subject in one ormore dosages ranging from about 0.01 μg to about 30 μg. In someembodiments wherein large animal models and humans are involved, thepriming composition and/or the boosting composition can be administeredinto the subject in one or more dosages ranging from about 0.1 μg toabout 100 μg. In some embodiments, suitable doses for small animalmodels range from about 5×10⁻⁵ μg/100 mg to about 0.15 μg/100 mg. Thisdose range is equivalent to about 0.01 μg to about 30 μg for a 20 gmouse. In some embodiments, the priming composition and/or the boostingcomposition is administered into the subject in multiple dosages ofabout 15 μg per dose for a 20 g mouse. In some embodiments, for largeanimal models, such as, for example humans, suitable dosages range fromabout 1.25×10⁻⁷ μg/100 mg to 1.25×10⁻⁴ μg/100 mg. This dose range isequivalent to about 0.1 μg to about 100 μg doses for an 80 kg host. Insome embodiments, the priming composition and/or the boostingcomposition is administered into the subject in multiple dosages ofabout 0.001 μg, about 0.01 μs, about 0.1 μg, about 1 μg, about 10 μg,about 100 μg, about 200 μg, about 300 μg of RNA per whole body dose informulated state. In some embodiments, the priming composition and/orthe boosting composition is administered into the subject in multipledosages of about 50 μg of RNA per whole body dose. In some embodiments,the priming composition and/or the boosting composition is administeredin gradually increasing dosages over time. In some embodiments, thepriming composition and/or the boosting composition is administered ingradually decreasing dosages over time.

In some embodiments of methods disclosed herein, immunization schedulecan be important. For example, a delayed boosting will be helpful inavoiding interference in the primary responses induced by the prime.Although closely spaced (e.g., 1-2 weeks) vaccine doses can cause arapid induction of immune response, in some embodiments, the responsemay be less persistent than when the same numbers of vaccine doses weregiven at longer intervals (e.g., 1-2 months). A minimal interval of 1-2weeks may also ensure optimal affinity maturation of memory B cells. Insome embodiments of the methods disclosed herein, the at least one doseof the priming composition and the boosting composition are administeredinto the subject at intervals of about 1 week, or 2, 3, 4, 5, 6, 7, or 8or 1-2 or 2-4 or 3-4 weeks. In some embodiments of the methods disclosedherein, the at least one dose of the priming composition and theboosting composition are administered into the subject at intervals ofabout 4 weeks. One of skill in the art will further appreciate that forany particular subject, specific dosage regimens can be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions. For example, doses may be adjusted based on clinicaleffects of the administered compositions such as toxic effects and/orlaboratory values. Dosage regimens can be adjusted to provide theoptimal desired effect. For example, as discussed above, a single dosecan be administered, several divided doses can be administered over timeor the dose can be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation. Determining appropriatedosages and regimens for administration of the compositions disclosedherein are well-known in the relevant art and would be understood to beencompassed by the skilled artisan once provided the teachings disclosedherein.

Thus, a person of skill in the art would appreciate, based upon thedisclosure provided herein, that the dose and dosing regimen is adjustedin accordance with methods well-known in the therapeutic arts. That is,the maximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a subject can alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the patient.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that can be provided to a patient in practicingthe present disclosure.

Administration of the priming and boosting compositions disclosed hereinmay be affected by any method that enables delivery of the compositionsto the site of action. These methods include oral routes, intraduodenalroutes, parenteral injection (comprising intravenous, subcutaneous,intramuscular, intravascular, or infusion), topical administration, andrectal administration. Infusions can be administered by drip, continuousinfusion, infusion pump, metering pump, depot formulation, or any othersuitable means. In some embodiments, at least one dose of the primingcomposition is administered intramuscularly to the subject. In someembodiments, at least one dose of the boosting composition isadministered intramuscularly to the subject.

In some embodiments, the at least one dose of boosting compositioncomprises different types of antigen comprising at least onecross-reactive epitope. In some embodiments, the method for heterologousprime-boost immunization disclosed herein intends to encompassimmunization regimens in which one of the multiple boosting compositionscomprises the same RNA replicon as used in the priming composition andthus a “homologous boost,” either of the same or different doses, aslong as at least one of the multiple administrations of the boostingcomposition comprises a RNA replicon that is different from that used inthe priming composition.

In some embodiments, the first antigen in the priming composition can bethe same antigen as the second antigen in the boosting composition. Insome embodiments, the first antigen and the second antigen have the sameamino acid sequence. In some embodiments, the amino acid sequence of thefirst antigen is a portion (for example, at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99%) of the amino acid sequence of the second antigen.In some embodiments, the first and the second antigens comprise aminoacid sequences that are homologous (for example substantially identical)to each other. The term “identical” or “percent identity” as used hereinin the context of two or more polymeric molecules, e.g., amino acidsequences of polypeptides, refers to the sequence similarity between thepolymeric molecules. Two amino acid sequences are homologous (e.g.,substantially identical) if there is a partial or complete identitybetween their sequences. For example, 80% identical means that 80% ofthe amino acids are identical when the two sequences are aligned formaximum matching. As such, the term “substantially identical” refers toa first amino acid which contains a sufficient or minimum number ofidentical or equivalent (e.g., with similar side chain) amino acids to asecond amino acid sequence such that the first and the second amino acidsequences have a common domain, such as an immunologically antigenicdeterminant (e.g., epitope). For example, the amino acid sequence of thefirst antigen can exhibit 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%,or a range between any two of these values, sequence identity to theamino acid sequence of the second antigen. In some embodiments, theamino acid sequence of the first antigen exhibits at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the amino acid sequence of the secondantigen. In some embodiments, the amino acid sequence of the firstantigen exhibits at least 80% sequence identity to the amino acidsequence of the second antigen. In some embodiments, the amino acidsequence of the first antigen exhibits at least 90% sequence identity tothe amino acid sequence of the second antigen. In some embodiments, theamino acid sequence of the first antigen exhibits at least 95% sequenceidentity to the amino acid sequence of the second antigen. In someembodiments, the amino acid sequence of the first antigen exhibits atleast 98% sequence identity to the amino acid sequence of the secondantigen. In some embodiments, the amino acid sequence of the firstantigen exhibits at least 99% sequence identity to the amino acidsequence of the second antigen. In some embodiments, the amino acidsequence of the first antigen exhibits at least 100% sequence identityto the amino acid sequence of the second antigen. In some embodiments,the amino acid sequence of the first antigen is identical to the aminoacid sequence of the second antigen.

As used herein, the terms, “identical” or percent “identity”, in thecontext of two or more nucleic acid sequences or polypeptide sequences,refer to two or more sequences or subsequences that are the same or havea specified percentage of amino acid residues or nucleotides that arethe same, when compared and aligned for maximum correspondence over acomparison window. Unless otherwise specified, the comparison window fora selected sequence, e.g., “SEQ ID NO: X” is the entire length of SEQ IDNO: X, and, e.g., the comparison window for “100 bp of SEQ ID NO: X” isthe stated 100 bp. The degree of amino acid or nucleic acid sequenceidentity can be determined by various computer programs for aligning thesequences to be compared based on designated program parameters. Forexample, sequences can be aligned and compared using the local homologyalgorithm of Smith & Waterman Adv. Appl. Math. 2:482-89, 1981, thehomology alignment algorithm of Needleman & Wunsch J. Mol. Biol.48:443-53, 1970, or the search for similarity method of Pearson & LipmanProc. Nat'l. Acad. Sci. USA 85:2444-48, 1988, and can be aligned andcompared based on visual inspection or can use computer programs for theanalysis (for example, GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-87, 1993). The smallest sum probability (P(N)), provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, preferably less than about 0.01,and more preferably less than about 0.001.

In some embodiments, the first and the second antigens comprise at leastone cross-reactive antigenic determinant. The term “epitope” or“antigenic determinant”, as used interchangeably herein, refers to theprimary, secondary, tertiary, or quaternary structure of an antigenicmolecule (e.g., a polypeptide) recognized by B cells (e.g., Blymphocytes) and the antibodies secreted by B cells. Epitopes can belinear or conformational. Generally, an epitope includes at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutiveamino acids in a unique spatial conformation. Encompassed by the term“epitope” and “antigenic determinant” are simple epitopes, whichcomprise only a few contiguous amino acid residues, as well as complexepitopes that encompass discontinuous amino acid residues. In somecases, complex epitopes comprise amino acid residues separated in theprimary sequence but in close proximity in the three-dimensional foldedstructure of an antigen. The term “cross-reactive antigenic determinant”“or cross-reactive epitope” refers to the ability of an antigenicdeterminant present on two or more antigen molecules (e.g.,polypeptides) to be bound by the same antibody. Furthermore, it is to beunderstood that the two or more molecules comprising the antigenicdeterminant capable of being bound by the same antibody can be, forexample, the same molecules or fragments thereof, variants of oneanother, or different molecules. By way of example with reference topolypeptides comprising an antigenic determinant capable of being boundby the same antibody, the polypeptides can have the same or a differentprimary amino acid sequence, however, the polypeptides each comprise anantigenic determinant (e.g., “cross-reactive”) that can be bound by thesame antibody. In some embodiments, the first and the second antigensinduce substantially the same immune response in the subject. In someembodiments, the term “substantially the same immune response” can referto, for example, where the concentration of antibodies induced againstthe first antigen is about the same, or at least about 75%, or at leastabout 80%, or at least about 90%, or at least about 95%, or at leastabout 99% of the concentration of antibodies induced against the secondantigen tested under the same conditions. In some embodiments, the firstand the second antigens induce the same immune response in the subject,e.g., the concentration of antibodies induced against the first antigenis identical to the concentration of antibodies induced against thesecond antigen tested under the same conditions.

In some embodiments, the term “substantially the same immune response”can refer to, for example, where the type of antibody profile inducedagainst the first antigen is about the same, or at least about 75%, orat least about 80%, or at least about 90%, or at least about 95%, or atleast about 99% identical to the type of antibody profile inducedagainst the second antigen tested under the same conditions. In someembodiments, the first and the second antigens induce the same immuneresponse in the subject, e.g., the type of antibody profile inducedagainst the first antigen is identical to the type of antibody profileinduced against the second antigen tested under the same conditions.

In some embodiments disclosed herein, the first and the second RNAreplicons are capable of activating an immune system of the subjectthrough different immunological mechanisms, e.g. differentially engagingor activating the immune system of a subject patient. For example, insome embodiments, the first RNA replicon can activate the immune systemof the subject through an immunological mechanism that is different fromone or more, or any, of the immunological mechanisms that the second RNAreplicon is capable of activating the immune system in the subject. Insome embodiments, each of the first and second RNA replicons mayindependently be capable of activating the immune system of the subjectthrough one, two, three, or more immunological mechanisms. In someembodiments, the first and second RNA replicons can activate the immunesystem through one, two, three, or more common immunological mechanisms;however, at least one of the immunological mechanisms utilized by thefirst RNA replicon is different from each of the immunologicalmechanisms utilized by the second RNA replicon. Non-limiting examples ofimmunological mechanisms through which the first and/or the secondreplicons can activate the immune system include (1) different activemechanisms of host cell immune evasion encoded by non-structuralproteins of a distinct or related replicon; (2) different passivemechanisms for the host cell immunity to recognize the replicon itself;and (3) co-encoding of immune modulating proteins that function todifferentially engage or activate the immune system during the first andsecond injection. In some embodiments of the disclosure, the at leastone of the two or more immunological mechanisms is selected from thegroup consisting of differential activation of protein kinase R (PKR),retinoic acid-inducible gene I (RIG-I), autophagy pathways, Toll-likereceptors (TLRs), stress granules, RNase R, and oligoadenylatesynthetases (OAS).

In some embodiments, at least one of the first and second RNA repliconsis a modified replicon. In some embodiments, the first and second RNAreplicons are derived from a positive-strand RNA virus. In someembodiments, at least one of the first and second RNA replicons isderived from a virus species belonging to a family selected from thegroup consisting of Togaviridae family, Flaviviridae family,Orthomyxoviridae family, Rhabdoviridae family, Arteroviridae family,Picornaviridae family, Astroviridae family, Coronaviridae family, andParamyxoviridae family. Accordingly, in some embodiments, at least oneof the first and second RNA replicons is derived from a negative-strandRNA virus. Suitable negative-strand RNA virus species include, but arenot limited to viral species of the families Orthomyxoviridae,Rhabdoviridae, and Paramyxoviridae. In some embodiments, at least one ofthe first and second RNA replicons is derived from a virus speciesbelonging to the Orthomyxoviridae family. In some embodiments, at leastone of the first and second RNA replicons is derived from a virusspecies belonging to a Orthomyxovirus genus selected from the groupconsisting of Influenza virus A, Influenza virus B, Influenza virus C,Influenza virus D, Isavirus, Thogotovirus and Quaranjavirus. In someembodiments, at least one of the first and second RNA replicons isderived from an Influenza virus. In some embodiments, at least one ofthe first and second RNA replicons is derived from an Influenza virus A.

In some embodiments, at least one of the first and second RNA repliconsis derived from a virus species belonging to the Rhabdoviridae family.In some embodiments, at least one of the first and second RNA repliconsis derived from a virus species belonging to a Rhabdovirus genusselected from the group consisting of Curiovirus, Cytorhabdovirus,Dichorhavirus, Ephemerovirus, Hapavirus, Ledantevirus, Lyssavirus,Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus,Sprivivirus, Sripuvirus, Tibrovirus, Tupavirus, Varicosavirus,Vesiculovirus. Non-limiting examples of preferred Rhabdovirus speciesinclude, but are not limited to, viral hemorrhagic septicemia virus(VHSV), vesicular stomatitis virus (VSV), and rabies virus (RABV).

In some embodiments, at least one of the first and second RNA repliconsis derived from a virus species belonging to the Paramyxoviridae family.In some embodiments, at least one of the first and second RNA repliconsis derived from a Paramyxovirus virus species belonging to thePneumovirinae subfamily or the Paramyxovirinae subfamily. In someembodiments, at least one of the first and second RNA replicons isderived from a virus species belonging to a Paramyxovirus genus selectedfrom the group consisting of Aquaparamyxovirus, Avulavirus, Ferlavirus,Henipavirus, Metapneumovirus, Morbillivirus, Pneumovirus, Respirovirus,and Rubulavirus. Non-limiting examples of preferred Paramyxovirusspecies include, but are not limited to, human respiratory syncytialvirus (hRSV, subgroup A), bovine respiratory syncytial virus (bRSV),human metapneumovirus (hMPV), bovine-human parainfluenza virus 3(b/hPIV3), human parainfluenza virus 1 (hPIV1), recombinant bovine-humanparainfluenza virus 3 (rB/HPIV3), Sendai virus (SeV), Andes virus(ANDV), Mumps virus (MuV), Simian virus 5 (SV5), and Measles virus(MeV).

In some embodiments, at least one of the first and second RNA repliconsis derived from a positive-strand virus species belonging to theTogaviridae family or Flaviviridae family. In some embodiments, at leastone of the first and second RNA replicons is derived from a virusspecies belonging to the Flaviviridae family such as, for example,viruses belonging to the genera Flavivirus and Pestivirus. Non-limitingexamples of viruses belonging to the genus Flavivirus include yellowfever virus (YFV), Dengue fever virus, Japanese encephalitis virus(JEV), West Nile virus (WNV) and Zika virus. In some embodiments, atleast one of the first and second RNA replicons is derived from yellowfever virus or Dengue fever virus. Virulent and avirulent flavivirusstrains are both suitable. Non-limiting examples of preferred flavivirusstrains include, but are not limited to, YFV (17D), DEN4 (814669 andderivatives), DEN2 (PDK-53), Kunjin virus (KUN), JEV (SA14-14-2), MurrayValley encephalitis virus (MVEV, with IRES attenuated), WNV (SCFV),Bovine viral diarrhea virus (BVDV) CP7, BVDV-SD1, BVDV-NADL, andclassical swine fever virus (CSFV).

In some embodiments, at least one of the first and second RNA repliconsis derived from a positive-strand virus species, for example a virusspecies belonging to the Alphavirus genus of the Togaviridae family. Insome embodiments, at least one of the first and second RNA replicons isderived from a positive-strand virus species belonging to theArterivirus genus of the Arteriviridae family. In some embodiments, atleast one of the first and second RNA replicons is derived from apositive-strand virus species belonging to the Arterivirus genus of theArteriviridae family and the other RNA replicon is derived from aspecies belonging to the Alphavirus genus of the Togaviridae family.

Alphaviruses

Alphavirus is a genus of genetically, structurally, and serologicallyrelated viruses of the group IV Togaviridae family which includes atleast 30 members, each having single stranded RNA genomes of positivepolarity enclosed in a nucleocapsid surrounded by an envelope containingviral spike proteins. Currently, the alphavirus genus comprises amongothers the Sindbis virus (SIN), the Semliki Forest virus (SFV), the RossRiver virus (RRV), Venezuelan equine encephalitis virus (VEEV), andEastern equine encephalitis virus (EEEV), which are all closely relatedand are able to infect various vertebrates such as mammals, rodents,fish, avian species, and larger mammals such as humans and horses aswell as invertebrates such as crustaceans and insects. Transmissionbetween species and individuals occurs mainly via mosquitoes making thealphaviruses a contributor to the collection of Arboviruses—orArthropod-Borne Viruses. For example, the Sindbis and the Semliki Forestviruses have been widely studied and the life cycle, mode ofreplication, etc., of these viruses are well characterized. For example,alphaviruses have been shown to replicate very efficiently in animalcells which makes them valuable as vectors for production of protein andnucleic acids in such cells.

Alphavirus particles are enveloped, have a 70 nm diameter, tend to bespherical (although slightly pleomorphic), and have an approximately 40nm isometric nucleocapsid. The Alphavirus genome is single-stranded RNAof positive polarity of approximately 11-12 kb in length, comprising a5′ cap, a 3′ poly-A tail, and two open reading frames with a first frameencoding the nonstructural proteins with enzymatic function and a secondframe encoding the viral structural proteins (e.g., the capsid proteinC, E1 glycoprotein, E2 glycoprotein, E3 protein and 6K protein).

The 5′ two-thirds of the alphavirus genome encodes a number ofnonstructural proteins necessary for transcription and replication ofviral RNA. These proteins are translated directly from the RNA andtogether with cellular proteins form the RNA-dependent RNA polymeraseessential for viral genome replication and transcription of subgenomicRNA. Four nonstructural proteins (nsP1-4) are produced as a singlepolyprotein and constitute the virus' replication machinery. Theprocessing of the polyprotein occurs in a highly regulated manner, withcleavage at the P2/3 junction influencing RNA template use during genomereplication. This site is located at the base of a narrow cleft and isnot readily accessible. Once cleaved, nsP3 creates a ring structure thatencircles nsP2. These two proteins have an extensive interface.Mutations in nsP2 that produce noncytopathic viruses or a temperaturesensitive phenotypes cluster at the P2/P3 interface region. P3 mutationsopposite the location of the nsP2 noncytopathic mutations preventefficient cleavage of P2/3. This in turn can affect RNA infectivityaltering viral RNA production levels.

The 3′ one-third of the genome comprises subgenomic RNA which serves asa template for translation of all the structural proteins required forforming viral particles: the core nucleocapsid protein C, and theenvelope proteins P62 and E1 that associate as a heterodimer. The viralmembrane-anchored surface glycoproteins are responsible for receptorrecognition and entry into target cells through membrane fusion. Thesubgenomic RNA is transcribed from the p26S subgenomic promoter presentat the 3′ end of the RNA sequence encoding the nsp4 protein. Theproteolytic maturation of P62 into E2 and E3 causes a change in theviral surface. Together the E1, E2, and sometimes E3, glycoprotein“spikes” form an E1/E2 dimer or an E1/E2/E3 trimer, where E2 extendsfrom the center to the vertices, E1 fills the space between thevertices, and E3, if present, is at the distal end of the spike. Uponexposure of the virus to the acidity of the endosome, E1 dissociatesfrom E2 to form an E1 homotrimer, which is necessary for the fusion stepto drive the cellular and viral membranes together. The alphaviralglycoprotein E1 is a class II viral fusion protein, which isstructurally different from the class I fusion proteins found ininfluenza virus and HIV. The E2 glycoprotein functions to interact withthe nucleocapsid through its cytoplasmic domain, while its ectodomain isresponsible for binding a cellular receptor. Most alphaviruses lose theperipheral protein E3, while in Semliki viruses it remains associatedwith the viral surface. In some embodiments the first and/or second RNAreplicons do not code for any viral structural protein, or do not codefor E1 or E2 or E3, or any one of them or any combination of them. Forexample when the RNA replicon is derived from an alphavirus it can notencode for E1 or E2 or E3, nor any combination or sub-combination ofthem.

Alphavirus replication is a membrane-associated process within the hostcell. In the first step of the infectious cycle, the 5′ end of thegenomic RNA is translated into a polyprotein (nsP1-4) with RNApolymerase activity that produces a negative strand complementary to thegenomic RNA. In a second step, the negative strand is used as a templatefor the production of two RNAs, respectively: (1) a positive genomic RNAcorresponding to the genome of the secondary viruses producing, bytranslation, other nsp proteins and acting as a genome for the virus;and (2) subgenomic RNA encoding the structural proteins of the virusforming the infectious particles. The positive genomic RNA/subgenomicRNA ratio is regulated by proteolytic autocleavage of the polyprotein tonsp 1, nsp 2, nsp 3 and nsp 4. In practice, the viral gene expressiontakes place in two phases. In a first phase, there is main synthesis ofpositive genomic strands and of negative strands. During the secondphase, the synthesis of subgenomic RNA is virtually exclusive, thusresulting in the production of large amount of structural protein.

In some embodiments disclosed herein, at least one of the first andsecond RNA replicons is derived from an alphavirus species. In someembodiments, the alphavirus RNA replicon is derived from an alphavirusbelonging to the VEEV/EEEV group, or the SF group, or the SIN group (forreview, see, e.g. Strauss and Strauss. Microbiol. Rev., 58:3 p 492-562,1994). Non-limiting examples of SF group alphaviruses include SemlikiForest virus, O'Nyong-Nyong virus, Ross River virus, Middelburg virus,Chikungunya virus, Barmah Forest virus, Getah virus, Mayaro virus,Sagiyama virus, Bebaru virus, and Una virus. Non-limiting examples ofSIN group alphaviruses include Sindbis virus, Girdwood S.A. virus, SouthAfrican Arbovirus No. 86, Ockelbo virus, Aura virus, Babanki virus,Whataroa virus, and Kyzylagach virus. Non-limiting examples of VEEV/EEEVgroup alphaviruses include Eastern equine encephalitis virus (EEEV),Western equine encephalitis virus (WEEV), Venezuelan equine encephalitisvirus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Semlikiforest virus (SFV), Pixuna virus (PIXV), Middleburg virus (MIDV),Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus(RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus(SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), and Una virus (UNAV).Virulent and avirulent alphavirus strains are both suitable for themethods and compositions disclosed herein. In some particularembodiments, the alphavirus RNA replicon is derived from a Sindbis virus(SIN), a Semliki Forest virus (SFV), a Ross River virus (RRV), aVenezuelan equine encephalitis virus (VEEV), or an Eastern equineencephalitis virus (EEEV). In some embodiments, the alphavirus RNAreplicon is derived from a Venezuelan equine encephalitis virus (VEEV).

In some embodiments, both of the first and second RNA replicons arederived from alphavirus species. In some embodiments, the first andsecond RNA replicons are derived from the same alphavirus species orfrom two different alphavirus species. In some embodiments, the firstRNA replicon is derived from an alphavirus and the second RNA repliconis derived from a non-alphavirus species. Non-limiting examples ofnon-alphavirus RNA replicons include RNA replicons derived from virusspecies belonging to a family selected from the group consisting ofTogaviridae family, Flaviviridae family, Orthomyxoviridae family,Rhabdoviridae family, Arteroviridae family, Picornaviridae family,Astroviridae family, Coronaviridae family, and Paramyxoviridae family.Accordingly, in some embodiments, the non-alphavirus RNA replicon isderived from a negative-strand RNA virus. Suitable negative-strand RNAvirus species include, but are not limited to viral species of thefamilies Orthomyxoviridae, Rhabdoviridae, and Paramyxoviridae. In someembodiments, the non-alphavirus RNA replicon is derived from anegative-strand RNA virus species belonging to the Orthomyxoviridaefamily. In some embodiments, the non-alphavirus RNA replicon is derivedfrom a virus species belonging to an Orthomyxovirus genus selected fromthe group consisting of Influenza virus A, Influenza virus B, Influenzavirus C, Influenza virus D, Isavirus, Thogotovirus and Quaranjavirus. Insome embodiments, the non-alphavirus RNA replicon is derived from anInfluenza virus. In some embodiments, the non-alphavirus RNA replicon isderived from an Influenza virus A.

In some embodiments, the non-alphavirus RNA replicon is derived from anegative-strand RNA virus species belonging to the Rhabdoviridae family.In some embodiments, the non-alphavirus RNA replicon is derived from avirus species belonging to a Rhabdovirus genus selected from the groupconsisting of Curiovirus, Cytorhabdovirus, Dichorhavirus, Ephemerovirus,Hapavirus, Ledantevirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus,Perhabdovirus, Sigmavirus, Sprivivirus, Sripuvirus, Tibrovirus,Tupavirus, Varicosavirus, Vesiculovirus. Non-limiting examples ofpreferred Rhabdovirus species include, but are not limited to, viralhemorrhagic septicemia virus (VHSV), vesicular stomatitis virus (VSV),and rabies virus (RABV).

In some embodiments, the non-alphavirus RNA replicon is derived from anegative-strand RNA virus species belonging to the Paramyxoviridaefamily. In some embodiments, the non-alphavirus RNA replicon is derivedfrom a Paramyxovirus virus species belonging to the Pneumovirinaesubfamily or the Paramyxovirinae subfamily. In some embodiments, thenon-alphavirus RNA replicon is derived from a virus species belonging toa Paramyxovirus genus selected from the group consisting ofAquaparamyxovirus, Avulavirus, Ferlavirus, Henipavirus, Metapneumovirus,Morbillivirus, Pneumovirus, Respirovirus, and Rubulavirus. Non-limitingexamples of preferred Paramyxovirus species include, but are not limitedto, human respiratory syncytial virus (hRSV, subgroup A), bovinerespiratory syncytial virus (bRSV), human metapneumovirus (hMPV),bovine-human parainfluenza virus 3 (b/hPIV3), human parainfluenza virus1 (hPIV1), recombinant bovine-human parainfluenza virus 3 (rB/HPIV3),Sendai virus (SeV), Andes virus (ANDV), Mumps virus (MuV), Simian virus5 (SV5), and Measles virus (MeV).

In some embodiments, the non-alphavirus RNA replicon is derived from apositive-strand virus species belonging to the Togaviridae family orFlaviviridae family. In some embodiments, the non-alphavirus RNAreplicon is derived from a virus species belonging to the Flaviviridaefamily such as, for example, viruses belonging to the genera Flavivirusand Pestivirus. Non-limiting examples of viruses belonging to the genusFlavivirus include yellow fever virus (YFV), Dengue fever virus,Japanese encephalitis virus (JEV), West Nile virus (WNV) and Zika virus.In some embodiments, at least one of the first and second RNA repliconsis derived from yellow fever virus or Dengue fever virus. Virulent andavirulent flavivirus strains are both suitable. Non-limiting examples ofpreferred flavivirus strains include, but are not limited to, YFV (17D),DEN4 (814669 and derivatives), DEN2 (PDK-53), Kunjin virus (KUN), JEV(SA14-14-2), Murray Valley encephalitis virus (MVEV, with IRESattenuated), WNV (SCFV), Bovine viral diarrhea virus (BVDV) CP7,BVDV-SD1, BVDV-NADL, and classical swine fever virus (CSFV).

In some embodiments, the non-alphavirus RNA replicon is derived from apositive-strand virus species belonging to the Arteriviridae family,which can be a virus of the genus Arterivirus. Suitable arterivirusspecies include, but are not limited to, species of Equine arteritisvirus (EAV), Porcine respiratory and reproductive syndrome virus(PRRSV), Lactate dehydrogenase elevating virus (LDV), Simian hemorrhagicfever virus (SHFV), and wobbly possum disease virus (WPDV).

In some embodiments, at least one of the first and second RNA repliconscomprises a modified 5′-UTR with one or more nucleotide substitutions atposition 1, 2, 4, or a combination thereof. In some embodiments, atleast one of the nucleotide substitutions is a nucleotide substitutionat position 1 of the modified 5′-UTR. In some embodiments, at least oneof the nucleotide substitutions is a nucleotide substitution at position2 of the modified 5′-UTR. In some embodiments, at least one of thenucleotide substitutions is a nucleotide substitution at position 4 ofthe modified 5′-UTR. In some embodiments, the nucleotide substitutionsat position 2 of the modified 5′-UTR is a U→G substitution. In someembodiments, the nucleotide substitution at position 2 of the modified5′-UTR is a U→A substitution. In some embodiments, the nucleotidesubstitution at position 2 of the modified 5′-UTR is a U→C substitution.

In some embodiments of the disclosure, a part or the entire codingsequence for one or more viral structural proteins is absent and/ormodified in the RNA replicon disclosed herein. Thus, in some particularembodiments, the RNA replicon as disclosed herein includes a modified5-′UTR and is devoid of at least a portion of a nucleic acid sequenceencoding one or more viral structural proteins, for example, devoid ofthe first one, two, three, four, five, six, seven, eight, nine, ten, ormore nucleotides of the nucleic acid sequence encoding the viralstructural proteins. In some embodiments, the modified RNA replicon canbe devoid of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, ormore of the sequence encoding one or more of the structural polypeptidesE1, E2, E3, 6K, and capsid protein C, or one or more other sequencesencoding structural polypeptides. In some embodiments, the modified RNAreplicon is devoid of a substantial portion of or the entire sequenceencoding one of or more of the structural polypeptides E1, E2, E3, 6K,and capsid protein C, or one or more other sequences encoding structuralpolypeptides. As used herein, a “substantial portion” of a nucleic acidsequence encoding a viral structural protein comprises enough of thenucleic acid sequence encoding the viral structural protein to affordputative identification of that protein, either by manual evaluation ofthe sequence by one skilled in the art, or by computer-automatedsequence comparison and identification using algorithms such as BLAST(see, for example, Karlin & Altschul, 1993, supra). In some embodiments,the modified RNA replicon is devoid of at least part of or of the entiresequence encoding one or more of the structural polypeptides E1, E2, E3,or of any combination or sub-combination of them. The modified RNAreplicon can also be devoid of at least a portion of or of the entiresequence of protein 6K, and/or capsid protein C.

Viral Capsid Enhancer Sequences

In some embodiments disclosed herein, at least one of the first andsecond RNA replicons is a modified alphavirus replicon comprising one ormore RNA stem-loops in a structural element of a viral capsid enhancer.

Some viruses have sequences capable of forming one or more stem-loopelements/structures which can be used, for example, in a heterologousviral genome for enhancing translation of a coding sequence locateddownstream thereto. For example, the subgenomic mRNA of Sindbis virushas a stable RNA hairpin loop located downstream of the wild type AUGinitiator codon for the virus capsid protein (e.g., capsid enhancer).This stem-loop RNA structure is often referred to as the Downstream LooP(or DLP motif). The DLP structure was first characterized in Sindbisvirus (SINV) 26S mRNA and also detected in Semliki Forest virus (SFV).Recently, similar DLP structures have been reported to be present in atleast 14 other members of the Alphavirus genus including New World(MAYV, UNAV, EEEV (NA), EEEV (SA), AURAV) and Old World (SV, SFV, BEBV,RRV, SAG, GETV, MIDV, CHIKV, ONNV) members. The predicted structures ofthese Alphavirus 26S mRNAs were constructed based on SHAPE (selective2′-hydroxyl acylation and primer extension) data (Toribio et al.,Nucleic Acids Res. May 19; 44(9):4368-80, 2016), the content of which ishereby incorporated by reference). In the case of Sindbis virus, the DLPmotif is found in the first ˜150 nucleotides of the Sindbis subgenomicRNA. The hairpin is located downstream of the Sindbis capsid AUGinitiation codon (AUG at nucleotide 50 of the Sindbis subgenomic RNA)and results in stalling a ribosome such that the correct capsid gene AUGis used to initiate translation. Because the hairpin causes ribosomes topause eIF2α is not required to support translation initiation. Withoutbeing bound by any particular theory, it is believed that placing theDLP motif upstream of a coding sequence for any gene of interest (GOI)typically results in a fusion-protein of N-terminal capsid amino acidsthat are encoded in the hairpin region to the GOI-encoded proteinbecause initiation occurs on the capsid AUG not the GOI AUG. Inaddition, unmodified RNA replicons are often sensitive to the initialinnate immune system state of cells they are introduced into. If thecells/individuals are in a highly active innate immune system state, theRNA replicon performance (e.g., replication and expression of a GOI) canbe negatively impacted. By engineering a DLP to control initiation ofprotein translation, particularly of non-structural proteins, the impactof the pre-existing activation state of the innate immune system toinfluence efficient RNA replicon replication is removed or lessened. Theresult is more uniform expression of the GOI that can impact vaccineefficacy or therapeutic impact of a treatment. Further informationregarding alphavirus DLP can be found in, for example, U.S. patentapplication Ser. No. 15/831,230. In some embodiments, the viral capsidenhancer comprises a downstream loop (DLP) motif of the virus species,and wherein the DLP motif comprises at least one of the one or more RNAstem-loops. For example, in some embodiments, the viral capsid enhancercomprises a nucleic acid sequence exhibiting at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to any one or more of SEQ ID NOs:2-9. In some embodiments, the viral capsid enhancer comprises a nucleicacid sequence exhibiting about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,100%, or a range between any two of these values, sequence identity toany one or more of SEQ ID NOs: 2-9. In some embodiments, the nucleicacid sequence exhibits at least 95% sequence identity to any one or moreof SEQ ID NOs: 2-9.

In some embodiments, either one or both of the first and second RNAreplicons is a modified alphavirus replicon comprising at least about50, about 75, about 100, about 150, about 200, about 300 or morenucleotides from the 5′ coding sequence for a viral capsid protein. Insome embodiments, the viral capsid enhancer is derived from a capsidgene of an alphavirus species selected from the group consisting ofEastern equine encephalitis virus (EEEV), Venezuelan equine encephalitisvirus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixunavirus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV),O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus(BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV),Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus(AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus(KYZV), Western equine encephalitis virus (WEEV), Highland J virus(HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), and Buggy Creekvirus (BCRV). In some particular embodiments, the viral capsid enhanceris derived from a capsid gene of a Sindbis virus species or a SemlikiForest virus species. In yet some particular embodiments, the viralcapsid enhancer is derived from a capsid gene of a Sindbis virusspecies. Additionally, one of ordinary skill in the art will appreciatethat modifications may be made in the 5′ coding sequences from the viralcapsid protein without substantially reducing its enhancing activities(see, e.g., Frolov et al., J. Virology 70:1182, 1994; Frolov et al., J.Virology 68:8111, 1994). Preferably, such mutations substantiallypreserve the RNA hairpin structure formed by the 5′ capsid codingsequences.

In some embodiments, the viral capsid enhancer sequence does not containall of the 5′ coding sequences of the viral capsid protein that areupstream of the hairpin structure. In some embodiments, the viral capsidenhancer sequence may encode all or part of the capsid protein.Accordingly, in some embodiments disclosed herein, the capsid enhancerregion will not encode the entire viral capsid protein. In someembodiments, the viral capsid enhancer sequence will encode an aminoterminal fragment from the viral capsid protein. In those embodiments inwhich an otherwise functional capsid is encoded by the capsid enhancersequence, it may be desirable to ablate the capsid autoproteaseactivity.

In some embodiments, the viral capsid enhancer sequence included in theRNA replicons of the disclosure may be of any other variant sequencesuch as, for example, a synthetic sequence or a heterologous sequence,that can form an RNA hairpin functionally or structurally equivalent toone or more of the RNA stem-loops predicted for a viral capsid enhancerand which can act to enhance translation of RNA sequences operablylinked downstream thereto (e.g., coding sequence for a gene ofinterest).

In some embodiments, at least one of the first and second RNA repliconsis a modified alphavirus replicon that includes the coding sequence forat least one, at least two, at least three, or at least fourheterologous non-structural proteins. In some embodiments, the modifiedalphavirus replicon includes the coding sequence for a heterologousnon-structural protein nsP3. In some embodiments, the heterologousnon-structural protein nsP3 is a Chikungunya virus (CHIKV) nsP3 or aSindbis virus (SINV) nsP3. In some embodiments, at least one of thefirst and second antigens is expressed under control of a 26S subgenomicpromoter or a variant thereof. In some embodiments, at least one of thefirst and second antigens is expressed under control of an alphavirus26S subgenomic promoter or a variant thereof. In some embodiments, the26S subgenomic promoter is a SINV 26S subgenomic promoter, RRV 26Ssubgenomic promoter, or a variant thereof.

Arteriviruses

The arteriviruses (Family Arteriviridae, Genus Arterivirus) encompass animportant group of enveloped, single-stranded, positive-sense RNAviruses which infect domestic and wild animals. Arteriviruses share asimilar genome organization and replication strategy to that of membersof the family Coronaviridae (genera Coronavirus and Torovirus), butdiffer considerably in their genetic complexity, genome length,biophysical properties, size, architecture, and structural proteincomposition of the viral particles (e.g., virion). Currently, theArterivirus genus is considered to include equine arteritis virus (EAV),porcine reproductive and respiratory syndrome virus (PRRSV), lactatedehydrogenase-elevating virus (LDV) of mice, simian hemorrhagic fevervirus (SHFV), and wobbly possum disease virus (WPDV). Recent studieshave reported that the newly identified wobbly possum disease virus(WPDV) also belongs to the Arterivirus genus.

A typical arterivirus genome varies between 12.7 and 15.7 kb in lengthbut their genome organization is relatively consistent with some minorvariations. The arterivirus genome is a polycistronic positive strandRNA, with 5′ and 3′ non-translated regions (NTRs) that flank an array of10-15 known ORFs. The large replicase ORFs 1a and 1b occupy the5′-proximal three-quarters of the genome, with the size of ORF1a beingmuch more variable than that of ORF1b. Translation of ORF1a producesreplicase polyprotein (pp) 1a, whereas ORF1b is expressed by −1programmed ribosomal frameshifting (PRF), which C-terminally extendspp1a into pp1ab. In addition, a short transframe ORF has been reportedto overlap the nsp2-coding region of ORF1a in the +1 frame and to beexpressed by −2 PRF. The 3′-proximal genome part has a compactorganization and contains 8 to 12 relatively small genes, most of whichoverlap with neighboring genes. These ORFs encode structural proteinsand are expressed from a 3′-co-terminal nested set of subgenomic mRNAs.The organization of these ORFs is conserved, but downstream of ORF1b,SHFV and all recently identified SHFV-like viruses contain three or fouradditional ORFs (˜1.6 kb) that may be derived from an ancientduplication of ORFs 2-4. Together with the size variation in ORF1a, thispresumed duplication explains the genome size differences amongarteriviruses.

With regard to equine arteritis virus (EAV), the wild-type EAV genome isapproximately 12.7 kb in size. The 5′ three fourths of the genome codesfor two large replicase proteins 1a and 1ab; the amino acid sequences ofthe two proteins are N-terminally identical but due to a ribosomalframeshift the amino acid sequence of the C-terminal region of 1ab isunique. The 3′ one quarter of the EAV genome codes for the virus'sstructural protein genes, all of which are expressed from subgenomicRNAs. The subgenomic RNAs form a nested set of 3′ co-terminal RNAs thatare generated via a discontinuous transcriptional mechanism. Thesubgenomic RNAs are made up of sequences that are not contiguous withthe genomic RNA. All of the EAV subgenomic RNAs share a common 5′ leadersequence (156 to 221 nucleotides in length) that is identical to thegenomic 5′ sequence. The leader and body parts of the subgenomic RNAsare connected by a conserved sequence termed atranscriptional-regulatory sequence (TRS). The TRS is found on the 3′end of the leader (leader TRS) as well as in the subgenomic promoterregions located upstream of each structural protein gene (body TRS).Subgenomic RNAs are generated as the negative strand replicationintermediate RNA is transcribed. As transcription occurs, thereplication complex pauses as it comes to each body TRS and then thenascent negative strand RNA becomes associated with the complementarypositive strand leader TRS where negative strand RNA transcriptioncontinues. This discontinuous transcription mechanism results insubgenomic RNA with both 5′ and 3′ EAV conserved sequences. The negativestrand subgenomic RNAs then become the template for production of thesubgenomic positive sense mRNA.

Infectious cDNA clones, representing the entire genome of EAV, have beenreported (van Dinten 1997; de Vries et al., 2000, 2001; Glaser et al.,1999) and they been used to study EAV RNA replication and transcriptionfor nearly two decades (van Marle 1999, van Marle 1999a, Molenkamp 2000,Molenkamp 2000a, Pasternak 2000, Tijms 2001, Pasternak 2001, Pasternak2003, Pasternak 2004, van den Born 2005, Beerens & Snijder 2007, Tijms2007, Kasteren 2013). In addition, infectious clones have been generatedthat contain the chloramphenicol acetyltransferase (CAT) gene insertedin place of ORF2 and ORF7 and CAT protein was shown to be expressed incells electroporated with those RNAs (van Dinten 1997, van Marle 1999).Modifications of the infectious clone via site directed mutagenesis anddeletion of the structural protein gene regions has been used todetermine the requirement for each structural gene in support of RNAreplication (Molenkamp 2000). The study reported by Molenkamp 2000concluded that the structural genes are not required to support RNAreplication. Analysis of sequence homology requirements for TRS activityin subgenomic RNA production was conducted and used to better define howdiscontinuous transcription mechanistically occurs (van Marle 1999,Pasternak 2000, Pasternak 2001, Pasternak 2003, van den Born 2005) anddefective interfering RNAs have been used to understand the minimalgenomic sequences required for replication and packaging of RNA intovirus particles (Molenkamp 2000a). Further information in this regardcan be found in, for example, U.S. patent application Ser. No.15/486,131, which is hereby incorporated by reference in its entirety.

In some embodiments disclosed herein, at least one of the first andsecond RNA replicons is derived from an arterivirus species. Suitablearterivirus species includes Equine arteritis virus (EAV), Porcinerespiratory and reproductive syndrome virus (PRRSV), Lactatedehydrogenase elevating virus (LDV), Simian hemorrhagic fever virus(SHFV), and wobbly possum disease virus (WPDV). In some embodimentsdisclosed herein, at least one of the first and second RNA replicons isderived from an arterivirus species selected from the group consistingof Equine arteritis virus (EAV), Porcine respiratory and reproductivesyndrome virus (PRRSV), Lactate dehydrogenase elevating virus (LDV), andSimian hemorrhagic fever virus (SHFV). In some embodiments, thearterivirus RNA replicon is derived from an Equine arteritis virus(EAV). Virulent and avirulent arterivirus strains are both suitable.Non-limiting examples of preferred arterivirus strains include, but arenot limited to, EAV-virulent Bucyrus strain (VBS), LDV-Plagemann, LDV-C,PRRSV-type 1, and PRRSV-type 2. Exemplary preferred EAV strains include,but are not limited to, EAV VB53, EAV ATCC VR-796, EAV HK25, EAV HK116,EAV ARVAC MLV, EAV Bucyrus strain (Ohio), modified EAV Bucyrus,avirulent strain CA95, Red Mile (Kentucky), 84KY-A1 (Kentucky),Wroclaw-2 (Poland), Bibuna (Switzerland), and Vienna (Australia).Non-limiting preferred examples of PRRSV strains include PRRSV LV4.2.1,PRRSV 16244B, PRRSV HB-1(sh)/2002, PRRSV HB-2(sh)/2002, PRRSV HN1, PRRSVSD 01-08, PRRSV SD0802, PRRSV SD0803, PRRSV VR2332. Non-limitingpreferred examples of SHFV strains and variants include SHFV variantsSHFV-krtg1 a and -krtg1b (SHFV-krtg1a/b), SHFVkrtg2a/b (GenBankaccession # JX473847 to JX473850), SHFV-LVR, the SHFV prototype variantLVR 42-0/M6941 (NC 003092), SHFV-krc1 and SHFVkrc2 from Kibale redcolobus (HQ845737 and HQ845738, respectively). Other non-limitingexamples of preferred arteriviruses include PRRSV-Lelystad, the European(type 1) type strain (M96262); PRRSVVR2332, the North American (type 2)type strain (U87392); EAV-Bucyrus (NC 002532); EAV-s3685 (GQ903794);LDV-P, the Plagemann strain (U15146); and LDV-C, the neurovirulent typeC strain (L13298).

In some embodiments, the first and second RNA replicons are derived fromthe same arterivirus species or from two different arterivirus species.In some embodiments, the first RNA replicon is derived from anarterivirus and the second RNA replicon is derived from anon-arterivirus species. In some embodiments, the first RNA replicon isderived from an arterivirus and the second RNA replicon is derived froman alphavirus. In some embodiments, the first RNA replicon is anunmodified RNA replicon derived from an arterivirus species. In someembodiments, the first RNA replicon is a modified RNA replicon derivedfrom an arterivirus species.

In some embodiments disclosed herein, the first RNA replicon is an RNAreplicon derived from an alphavirus species and the second RNA repliconis an RNA replicon derived from an arterivirus species. In certainembodiments, the first RNA replicon is an unmodified RNA repliconderived from an alphavirus species. In other embodiments, the first RNAreplicon is a modified RNA replicon derived from an alphavirus species.

In some embodiments disclosed herein, the methods of the disclosurefurther include one or more subsequent boosting administrations. In someembodiments, the methods of the disclosure further include at least 2,at least 3, at least 4, at least 5, or at least 10 consecutive boostingadministrations or any number administration therebetween. In someembodiments, the subsequent boosting administrations are performed ingradually increasing dosages over time. In some embodiments, thesubsequent boosting administrations are performed in graduallydecreasing dosages over time.

In some embodiments, one or more of the priming composition and theboosting composition further comprises a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutically-acceptable carrier”means a carrier that is useful in preparing a pharmaceutical compositionor formulation that is generally safe, non-toxic, and neitherbiologically nor otherwise undesirable, and includes a carrier that isacceptable for veterinary use as well as human pharmaceutical use. Insome embodiments, a pharmaceutically acceptable carrier is as simple aswater, but it can also include, for example, a solution of physiologicalsalt concentration or saline. It can also be a lipid nanoparticle.Suitable materials include polyamidoamine (PAMAM) C12 dendrimers, and/or1,2-dimyristoiyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]. Polyethleneimine (PEI) and/or G5 and G9 NH2 PAMAMdendrimers can also be used. In some embodiments, a pharmaceuticallyacceptable carrier can be, or may include, stabilizers, diluents andbuffers. Suitable stabilizers are for example SPGA, carbohydrates (suchas dried milk, serum albumin or casein) or degradation products thereof.Suitable buffers are for example alkali metal phosphates. Diluentsinclude water, aqueous buffers (such as buffered saline), alcohols andpolyols (such as glycerol). For administration to animals or humans, thecomposition according to the present application can be given inter aliaparenterally, intranasally, by spraying, intradermally, subcutaneously,orally, by aerosol or intramuscularly.

In some embodiments, the first and the second RNA replicon eachcomprises at least one expression cassette comprising a promoteroperably linked to a coding sequence for a gene of interest (GOI). Asused herein, the term “expression cassette” refers to a construct ofgenetic material that contains coding sequences for a protein orfunctional RNA operably linked to expression control elements, such as apromoter, with enough regulatory information to direct propertranscription and/or translation of the coding sequences in a recipientcell, in vivo and/or ex vivo.

The term “operably linked”, as used herein, denotes a functional linkagebetween two or more sequences. For example, an operable linkage betweena polynucleotide of interest and a regulatory sequence (for example, apromoter) is a functional link that allows for expression of thepolynucleotide of interest. In this sense, the term “operably linked”refers to the positioning of a regulatory region and a coding sequenceto be transcribed so that the regulatory region is effective forregulating transcription or translation of the coding sequence ofinterest. In some embodiments disclosed herein, the term “operablylinked” denotes a configuration in which a regulatory sequence is placedat an appropriate position relative to a sequence that encodes apolypeptide or functional RNA such that the control sequence directs orregulates the expression or cellular localization of the mRNA encodingthe polypeptide, the polypeptide, and/or the functional RNA. Thus, apromoter is in operable linkage with a nucleic acid sequence if it canmediate transcription of the nucleic acid sequence. Operably linkedelements may be contiguous or non-contiguous.

Techniques for operably linking two or more sequences of DNA togetherare familiar to one of skill in the art, and such techniques have beendescribed in a number of texts for standard molecular biologicalmanipulation (see, for example, Maniatis et al., “Molecular Cloning: ALaboratory Manual” 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and Gibson et al., Nature Methods 6:343-45, 2009).

In some embodiments disclosed herein, the RNA replicons disclosed hereincan include more than one expression cassette. In principle, the RNAreplicons disclosed herein can generally include any number ofexpression cassettes. In some particular embodiments, the RNA repliconscomprise at least two, three, four, five, or six expression cassettes.In some embodiments, at least one of the one or more expressioncassettes is operably positioned downstream to a transcriptionalregulatory sequence (TRS) of an arterivirus RNA replicon, wherein theTRS is selected from the group consisting of TRS1, TRS2, TRS3, TRS4,TRS5, TRS6, and TRS7. In some particular embodiments, at least one ofthe one or more expression cassettes is operably positioned downstreamof the TRS7 of an arterivirus RNA replicon.

In some embodiments, the coding sequence of the GOI is optimized forexpression at a level higher than the expression level of a referencecoding sequence. In some embodiments, the reference coding sequence is asequence that has not been optimized. In some embodiments, theoptimization of the GOI coding sequence can include codon optimization.With respect to codon-optimization of nucleotide sequences, degeneracyof the genetic code provides the possibility to substitute at least onebase of the protein encoding sequence of a gene with a different basewithout causing the amino acid sequence of the polypeptide produced fromthe gene to be changed. Hence, the polynucleotides of the presentapplication may also have any base sequence that has been changed fromany polynucleotide sequence described herein by substitution inaccordance with degeneracy of the genetic code. References describingcodon usage are readily publicly available. In some further embodimentsof the disclosure, polynucleotide sequence variants can be produced fora variety of reasons, e.g., to optimize codon expression for aparticular host (e.g., changing codons in the arterivirus mRNA to thosepreferred by other organisms such as human, hamster, mice, or monkey).

In some embodiments disclosed herein, the GOI can encode an amino acidsequence of a polypeptide. The polypeptide can generally be anypolypeptide, and can be, for example a therapeutic polypeptide, aprophylactic polypeptide, a diagnostic polypeptide, a nutraceuticalpolypeptide, an industrial enzyme, and a reporter polypeptide. In someembodiments, the GOI encodes a polypeptide selected from the groupconsisting of an antibody, an antigen, an immune modulator, and acytokine. In some embodiments, the GOI encodes a polypeptide selectedfrom the group consisting of a therapeutic polypeptide, a prophylacticpolypeptide, a diagnostic polypeptide, a nutraceutical polypeptide, anindustrial enzyme, and a reporter polypeptide.

In some embodiments, the RNA replicons disclosed herein further comprisea coding sequence for a proteolytic cleavage site operably linkeddownstream to the third nucleotide sequence and upstream to the codingsequence for the GOI. Generally, any proteolytic cleavage site known inthe art can be incorporated into the polynucleotides and RNA repliconsof the disclosure and can be, for example, proteolytic cleavagesequences that are cleaved post-production by a protease. Furthersuitable proteolytic cleavage sites also include proteolytic cleavagesequences that can be cleaved following addition of an externalprotease. In some embodiments, RNA replicons disclosed herein furthercomprise a coding sequence for an autoprotease peptide operably linkeddownstream to the third nucleotide sequence and upstream to the codingsequence for the GOI. As used herein the term “autoprotease” refers to a“self-cleaving” peptide that possesses autoproteolytic activity and iscapable of cleaving itself from a larger polypeptide moiety. Firstidentified in the foot-and-mouth disease virus (FMDV), which is a memberof the picornavirus group, several autoproteases have been subsequentlyidentified such as, for example, “2A like” peptides from equine rhinitisA virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus(T2A), and their activities in proteolytic cleavage have been shown invarious in vitro and in vivo eukaryotic systems. As such, the concept ofautoproteases is available to one of skill in the art with manynaturally occurring autoprotease systems having been identified.Well-studied autoprotease systems include, but are not limited to, viralproteases, developmental proteins (e.g. HetR, Hedgehog proteins), RumAautoprotease domain, UmuD, etc.). Non-limiting examples of autoproteasepeptides suitable for the compositions and methods of the presentdisclosure include the peptide sequences from porcine teschovirus-1 2A(P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an EquineRhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), acytoplasmic polyhedrosis virus 2a (BmCPV2A), a Flacherie Virus 2A(BmIFV2A), or a combination thereof.

Compositions of the Disclosure

Some embodiments disclosed herein relate to a composition whichincludes: a priming composition comprising a first RNA replicon whichencodes a first antigen; and a boosting composition comprising a secondRNA replicon which encodes a second antigen, wherein the first andsecond RNA replicons are different from each other. In some embodiments,amino acid sequences of the first and the second antigens are homologousto each other. In some embodiments, the first and the second antigensare identical to each other. In some embodiments, the first and thesecond antigens comprise at least one cross-reactive antigenicdeterminant. In some embodiments, the composition is for inducing animmune response in a subject. In some embodiments, the first and thesecond antigens induce substantially the same immune response in thesubject. The composition can be, for example, a prophylactic compositionor a pharmaceutical composition comprising a pharmaceutically acceptablecarrier, or a mixture thereof. In some embodiments, the compositions ofthe present application can be used as a vaccine.

Some embodiments disclosed herein relate to a composition whichincludes: a first nucleic acid sequence encoding a first RNA repliconwhich encodes a first antigen; and a second nucleic acid sequenceencoding a second RNA replicon which encodes a second antigen, whereinthe first and second RNA replicons are different from each other,wherein the first replicon and the second replicon comprises at leastone expression cassette comprising a promoter operably linked to acoding sequence for a molecule of interest. In some embodiments, aminoacid sequences of the first and the second antigens are homologous toeach other. In some embodiments, the first and the second antigens areidentical to each other. In some embodiments, the first and the secondantigens comprise at least one cross-reactive antigenic determinant. Insome embodiments, the amino acid sequence of the first antigen exhibitsat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe amino acid sequence of the second antigen. In some embodiments, thecomposition is for producing a molecule of interest. In someembodiments, the molecule of interest is a polypeptide. The polypeptidecan generally be any polypeptide, and can be, for example a therapeuticpolypeptide, a prophylactic polypeptide, a diagnostic polypeptide, anutraceutical polypeptide, an industrial enzyme, and a reporterpolypeptide. In some embodiments, the molecule of interest is apolypeptide selected from the group consisting of an antibody, anantigen, an immune modulator, and a cytokine. In some embodiments, themolecule of interest is a polypeptide selected from the group consistingof a therapeutic polypeptide, a prophylactic polypeptide, a diagnosticpolypeptide, a nutraceutical polypeptide, an industrial enzyme, and areporter polypeptide.

Methods for Producing Molecules of Interest

The compositions and methods of the present disclosure can be used toproduce (e.g., express) a molecule of interest such as, e.g., apolypeptide, encoded in an open reading frame of a gene of interest(GOI) as disclosed herein. Thus, the present application furtherprovides compositions and methods for producing a molecule of interestsuch as, e.g., a polypeptide. Further information in this regard can befound in, for example, U.S. patent application Ser. Nos. 15/486,131;15/723,658, and 15/831,230.

Accordingly, some embodiments relate to methods for producing apolypeptide of interest in a subject, including sequentiallyadministering to the subject the first and the second RNA repliconsaccording to any one of the aspects and embodiments.

The methods and compositions disclosed herein can be used, for example,with subjects that are important or interesting for aquaculture,agriculture, animal husbandry, and/or for therapeutic and medicinalapplications, including production of polypeptides used in themanufacturing of vaccines, pharmaceutical products, industrial products,chemicals, and the like. In some embodiments, the compositions andmethods disclosed herein can be used with subjects that are naturalhosts of alphaviruses, such as rodents, mice, fish, birds, and largermammals such as humans, horses, pig, monkey, and apes as well asinvertebrates. Particularly preferred species, in some embodiments ofthe application, are vertebrate animal species and invertebrate animalspecies. In principle, any animal species can be generally used and canbe, for example, mammalian species such as human, horse, pig, primate,mouse, ferret, rat, cotton rat, cattle, swine, sheep, rabbit, cat, dog,goat, donkey, hamster, or buffalo. In some embodiments, the subject isan avian species, a crustacean species, or a fish species. In someembodiments, the avian species is an avian species for food consumption.Non-limiting examples of suitable avian species include chicken, duck,goose, turkey, ostrich, emu, quail, pigeon, swan, peafowl, pheasant,partridge, and guinea fowl. The term “crustacean” as used hereinincludes all crustacean species, for example those commonly referred toas “shrimp,” “lobsters,” “crawfish,” and “crabs,” such as Penaeus,Litopenaeus, Marsupenaeus, Fenneropenaeus, and Farfantepenaeus. In someembodiments, the crustacean species are shrimp species, particularlythose that are raised in aquaculture such as Litopenaeus vannamei,Penaeus vannamei, Penaeus styllirostris, Penaeus monodon, Pandalusborealis, Acetes japonicas, Trachysalambria curvirostris, andFenneropenaeus chinensis. In some embodiments, the fish are ornamentalfish or fish species used in aquaculture for consumption such as, eel,salmon, trout, carp, catfish, bass, and tilapia. In some embodiments,the fish species is in the Salmonidae family.

Techniques for transforming or transfecting a wide variety of theabove-mentioned subjects are known in the art and described in thetechnical and scientific literature.

All publications and patent applications mentioned in this disclosureare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

It will be clearly understood that, although a number of informationsources, including scientific journal articles, patent documents, andtextbooks, are referred to herein; this reference does not constitute anadmission that any of these documents forms part of the common generalknowledge in the art.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and alternativeswill be apparent to those of skill in the art upon review of thisdisclosure and are to be included within the spirit and purview of thisapplication.

EXAMPLES

Additional alternatives are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1 Increased Immune Response Following Heterologous Prime-BoostUsing Different RNA Replicons

This Example summarizes the experiments illustrating the induction of animmune response following heterologous prime-boost immunizationperformed with RNA replicons which activate an immune system of asubject through immunologically distinct mechanisms. As described above,heterologous prime-boost immunization is believed to generate enhancedimmune responses through (1) avoidance of anti-vector immunity, and (2)differential and synergistic activation of the immune response. Whileheterologous prime-boosts schedules have demonstrated efficacy usingdistinct platforms for delivery, this approach has been unavailablethrough the rational engineering of replicons. To date, replicons thatdifferentially engage the immune system have not been employed toimprove either T or B cell responses. In addition, the use of twodistinct systems to avoid anti-vector responses against replicons thatencode for a therapeutic protein has not been previously possible as amethod to enhance the magnitude or durability of protein expression.

Described herein is the use of two different fully synthetic repliconsystems as a means of enhancing the immune response in a heterologousprime-boost format. Although using two different systems forheterologous-prime boost has been effective for other vaccines due toavoidance of anti-vector immunity and differential activation of theimmune system, this has not formerly been possible or demonstrated usingreplicons. The reasons for this are two-fold. First, alphavirusreplicons are the only replicon system available currently in use, andthe novel engineering of EAV allowed for this method of immunization orprotein administration. Second, the lack of rational engineering withinthe same viral family to significantly alter the mechanism of immuneactivation between two different replicons to drive differential immuneresponses in a heterologous prime-boost format has not beendemonstrated.

In the experiments described in this Example, RNA replicons derived fromtwo different viruses: an arteritis virus (Equine arteritis virus—EAV)and an alphavirus (Venezuelan equine encephalitis virus—VEEV), were usedas representative of the heterologous RNA replicon prime-boost approach.Recombinant EAV-based and VEEV-based RNA replicons have been designedand subsequently used to vaccinate mice in a heterologous prime-boostvaccination regime. As described in more detail below, these recombinantRNA replicons have been tested in vivo in mouse models and demonstrateda differentiated and enhanced immune response in comparison with controlanimals receiving a homologous prime-boost regime. For example,Applicant has demonstrated in saline formulations that a heterologousprime-boost regime produces superior T cell response following aboosting step when compared to EAV-EAV immunization or VEEV-VEEVimmunization using a hemagglutinin (HA) antigen derived from InfluenzaA/Vietnam/1203/2003 (H5N1) strain.

To analyze the effect of heterologous prime-boosts on the immunogenicityof replicons, mice were immunized with combinations of either EAVreplicon or a VEE replicon containing a Downstream LooP sequence (or DLPmotif). Each replicon includes coding sequences for hemagglutinin (HA)from Influenza A/Vietnam/1203/2004 (H5N1). In these experiments, BALB/cmice were immunized with a dose of 15 μg formulated in saline andinjected intramuscularly at intervals of 4 weeks. Fourteen daysfollowing the final injection, spleens and serum were collected toanalyze the immune responses to HA. A summary of the results of theseexperiments is presented in FIGS. 2A-2B.

As shown in FIG. 2A, splenocytes were stimulated with conserved T cellepitope (H-2 Kd: IYSTVASSL; SEQ ID NO: 1) and revealed a significantincrease in IFN-γ-secreting CD8+ T cells in the group receiving aheterologous prime-boost regimen, where the priming composition includesan EAV replicon and the boosting composition includes a VEEV replicon,when compared to either homologously-primed group or single-dose group.

The above observation differed from animals that received a heterologousprime-boost regimen where the priming composition included a VEEVreplicon and the boosting composition included an EAV replicon,demonstrating the differential effects of a heterologous prime-boostregime compared with a homologous prime-boost regimen. Without beingbound by any particular theory, one possible explanation for theincreased CD8+ T cell response in the heterologous EAV-VEEVadministration group versus the homologous VEEV-VEEV group is adiminished anti-vector immunity to the viral non-structural proteinsencoded by the replicon. As discussed above, anti-vector immunity wouldresult in a more rapid clearance of cells expressing the replicon andthus result in restriction of the expressed antigen at boost.

The data presented above also suggests that the order of administrationof each replicon has an effect on the T cell responses generated.Specifically, a heterologous prime-boost schedule with a VEEV-basedreplicon first followed by a EAV-based replicon did not generate thesame frequency of IFNγ+ antigen-specific CD8+ T cells than, aheterologous prime-boost regimen with a EAV priming first followed by aVEEV boost. In agreement with the above observation, the order ofadministration has also been shown to give differential T cell responsesin other heterologous prime-boost vaccine model systems. For example,for protection against malaria using viral-based vectors, priming withan Adenovirus-based vector encoding the malarial antigen ME.TRAP,followed by a boost with a modified Vaccinia Ankara based vectorencoding the same antigen resulted in better T cell memory responses andenhanced protection than the reverse order of administration.

As shown in FIG. 2B, B cell responses in various heterologousprime-boost regimens were also examined. Serum from animals werecollected at fourteen (14) days post the final injection and assessedfor HA-specific total IgG responses. In contrast to elevated CD8+ T cellresponses, B cell responses from animals that received a heterologousEAV-VEEV prime-boost regimen showed a marginal and slightly decreasedlevel of antigen-specific total IgG when compared to the VEEV-VEEVhomologous regimen group. However, B cell responses observed in bothheterologous prime-boost groups (i.e. EAV-VEEV and VEEV-EAV) weresignificantly higher than animals receiving a single dose. In thismanner, heterologous prime-boost can be used to elicit B cell responseswith significantly improved effector CD8+ T cell responses.

It was unexpected that T cell responses at 14 days post-boost wereimproved using two different replicons in contrast to a homologousprime-boost regime. However, any differences in immune responses wereconceptually unexpected since this has not been attempted previously.Specifically, it was unexpected that a VEEV replicon prime followed byan EAV replicon boost yielded an inferior T cell response to an EAVreplicon prime and VEEV replicon boost. Furthermore, it was alsounexpected that heterologous prime-boost enhanced both T and B cellresponses when compared to EAV-EAV replicon homologous prime-boost, butonly superior T cell responses when compared with an VEEV-VEEV repliconhomologous prime-boost. Finally, in contrast to other demonstrations ofheterologous prime-boosts, the observation that mRNAs capable ofself-amplification, which are chemically similar, could differentiallyaffect downstream immune responses following administration is alsounexpected.

In particular, since a prime immunization with an EAV-based repliconfollowed by a boost immunization with an alphavirus-based replicondemonstrated the best T cell responses, additional experiments are alsoperformed to prime the immune system with an EAV-based replicon followedby a boost immunization using each of the alphavirus-based repliconslisted below in Table 1.

TABLE 1 Non-limiting exemplary combinations of heterologous prime-boostregimens of the present disclosure. Prime Boost 1 EAV replicon nt2 pointmutant of the alphavirus replicon* 2 nt2 point mutant of the alphavirusreplicon EAV replicon 3 DLP motif-containing alphavirus replicon nt2point mutant of the alphavirus replicon 4 nt2 point mutant of thealphavirus replicon DLP motif-containing alphavirus replicon 5 EAVreplicon CHIKV nsP3 variant alphavirus replicon 6 EAV replicon SINV nsP3variant alphavirus replicon 7 EAV replicon RRV 26S promoter variantalphavirus 8 EAV replicon SINV 26S promoter variant alphavirus 9 DLPmotif-containing alphavirus replicon CHIKV nsP3 variant alphavirusreplicon 10 DLP motif-containing alphavirus replicon SINV nsP3 variantalphavirus replicon 11 CHIKV nsP3 variant alphavirus replicon DLPmotif-containing alphavirus replicon 12 SINV nsP3 variant alphavirusreplicon DLP motif-containing alphavirus replicon 13 RRV 26S promotervariant alphavirus DLP motif-containing alphavirus replicon 14 DLPmotif-containing alphavirus replicon RRV 26S promoter variant alphavirus15 SINV 26S promoter variant alphavirus DLP motif-containing alphavirusreplicon 16 DLP motif-containing alphavirus replicon SINV 26S promotervariant alphavirus 17 WT alphavirus replicon** DLP motif-containingalphavirus replicon 18 DLP motif-containing alphavirus replicon WTalphavirus replicon *alphavirus replicon comprising a modified 5′-UTRwith a nucleotide substitution at position 2. **Wild-type/unmodifiedalphavirus replicon.

Additional experiments are also performed to demonstrate the following:(1) superiority of the immune responses of EAV-VEEV or VEEV-EAV eitherin saline or LNP (cationic lipid nanoparticle) formulations, (2)superiority of two VEEV replicons with immunological mechanisms ofimmune activation, and (3) superiority of heterologous prime-boost in atherapeutic setting.

While particular alternatives of the present disclosure have beendisclosed, it is to be understood that various modifications andcombinations are possible and are contemplated within the true spiritand scope of the appended claims. There is no intention, therefore, oflimitations to the exact abstract and disclosure herein presented.

What is claimed is:
 1. A method for inducing an immune response in asubject, comprising: administering to the subject at least one dose of apriming composition comprising a first RNA replicon which encodes afirst antigen; and subsequently administering to the subject at leastone dose of a boosting composition comprising a second RNA repliconwhich encodes a second antigen, wherein the first and second RNAreplicons are different from each other.
 2. The method of claim 1,wherein the first and the second antigens comprise at least onecross-reactive antigenic determinant.
 3. The method of claim 1, whereinthe first RNA replicon activates an immune system of the subject throughat least one immunological mechanism that is different from animmunological mechanism by which the second RNA replicon activates theimmune system.
 4. The method of claim 1, wherein at least one of thefirst and second RNA replicons is derived from a positive-strand RNAvirus.
 5. The method of claim 4, wherein at least one of the first andsecond RNA replicons is derived from a virus species belonging to afamily selected from the group consisting of Togaviridae family,Flaviviridae family, Orthomyxoviridae family, Rhabdoviridae family,Arteroviridae family, Picornaviridae family, Astroviridae family,Coronaviridae family, and Paramyxoviridae family.
 6. The method of claim1, wherein the first RNA replicon is derived from a non-alphavirus andthe second RNA replicon is derived from an alphavirus species.
 7. Themethod of claim 6, wherein the first RNA replicon is derived from anArterivirus.
 8. The method of claim 6, wherein the second RNA repliconis derived from an alphavirus species selected from the group consistingof Eastern equine encephalitis virus (EEEV), Venezuelan equineencephalitis virus (VEEV), Everglades virus (EVEY), Mucambo virus(MUCV), Semliki forest virus (SFV), Pixuna virus (PIXV), Middleburgvirus (MIDY), Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV),Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET),Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MA YV), Unavirus (UNA V), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus(WHAV), Babanki virus (BABY), Kyzylagach virus (KYZV), Western equineencephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus(FMV), Ndumu virus (NDUV), Salmonid alphavirus (SAV), and Buggy Creekvirus (BCRV).
 9. The method of claim 8 wherein the second RNA repliconis derived from an Arterivirus species selected from the groupconsisting of: equine arteritis virus (EAV), porcine reproductive andrespiratory syndrome virus (PRRSV), lactate dehydrogenase elevatingvirus (LDV) of mice, simian hemorrhagic fever virus (SHFV), and wobblypossum disease virus (WPDV).
 10. The method of claim 1, wherein at leastone of the first and second RNA replicons comprises a modified 5′-UTRwith one or more nucleotide substitutions at position 1, 2, 4, or acombination thereof.
 11. The method of claim 10, wherein at least one ofthe one or more nucleotide substitutions is a nucleotide substitution atposition 2 of the modified 5′-UTR.
 12. The method of claim 11, whereinthe nucleotide substitution at position 2 of the modified 5′-UTR is aU->G substitution.
 13. The method of claim 1, wherein at least one ofthe first and second RNA replicons is a modified RNA replicon comprisinga modified 5′-UTR and is devoid of at least a portion of a nucleic acidsequence encoding one or more viral structural proteins.
 14. The methodof claim 1, wherein at least one of the first and second RNA repliconsis a modified alphavirus replicon comprising one or more RNA stem-loopsin a structural element of a viral capsid enhancer or a variant thereof.15. The method of claim 1, wherein at least one of the first and secondRNA replicons is a modified alphavirus replicon comprising a codingsequence for a heterologous non-structural protein nsP3.
 16. The methodof claim 15, wherein the heterologous non-structural protein nsP3 is aChikungunya virus (CHIKV) nsP3, a Sindbis virus (SINV) nsP3, or avariant thereof.
 17. The method of claim 1, wherein at least one of thefirst and second antigens is expressed under control of a 26S subgenomic promoter or a variant thereof.
 18. The method of claim 17,wherein the 26S subgenomic promoter is a SINV 26S subgenomic promoter, aRRV 26S subgenomic promoter, or a variant thereof.
 19. The method ofclaim 1, wherein the first RNA replicon is derived from an arterivirusspecies and the second RNA replicon is derived from a non-arterivirusspecies.
 20. The method of claim 19, wherein the arterivirus species isselected from the group consisting of Equine arteritis virus (EAV),Porcine respiratory and reproductive syndrome virus (PRRSV), Lactatedehydrogenase elevating virus (LDV), and Simian hemorrhagic fever virus(SHFV).
 21. The method of claim 20, wherein the second RNA replicon isderived from an alphavirus.
 22. The method of claim 21 wherein the firstRNA replicon is derived from EAV and the second RNA replicon is derivedfrom an alphavirus.
 23. The method of claim 22 wherein the alphavirus isderived from VEEV.
 24. The method of claim 19 wherein the first RNAreplicon and the second RNA replicon each comprise a sequence encoding agene of interest.
 25. The method of claim 24 wherein the gene ofinterest encodes a polypeptide that is an antigenic determinant to thesubject.
 26. The method of claim 19, wherein the method comprisesadministering two or more doses of the boosting composition to thesubject.
 27. The method of claim 19, wherein one or more of the primingcomposition and the boosting composition comprises a pharmaceuticallyacceptable carrier.
 28. The method of claim 19, wherein the subject isan avian species, a crustacean species, or a fish species.
 29. Themethod of claim 19, wherein the subject is a mammal.
 30. The method ofclaim 19, wherein the subject is an aquatic animal or an avian species.31. A method for delivering two RNA replicons into a subject, comprisingadministering to the subject a first nucleic acid sequence encoding afirst RNA replicon which encodes a first antigen; and subsequentlyadministering to the subject, a second nucleic acid sequence encoding asecond RNA replicon which encodes a second antigen, wherein the firstand second RNA replicons are different from each other.
 32. Acomposition comprising: a priming composition comprising a first RNAreplicon which encodes a first antigen; and a boosting compositioncomprising a second RNA replicon which encodes a second antigen whereinthe first and second RNA replicons are different from each other.
 33. Acomposition comprising: a first nucleic acid sequence encoding a firstRNA replicon which encodes a first antigen; and a second nucleic acidsequence encoding a second RNA replicon which encodes a second antigen,wherein the first and second RNA replicons are different from eachother, wherein the first replicon and/or the second replicon comprisesat least one expression cassettes comprising a promoter operably linkedto a coding sequence for a molecule of interest.